Method of producing herpes simplex virus amplicons, resulting amplicons, and their use

ABSTRACT

The present invention relates to a method for producing herpes simplex virus (HSV) amplicon particles which includes co-transfecting a host cell with the following: (i) an amplicon vector comprising an HSV origin of replication, an HSV cleavage/packaging signal, and a heterologous transgene expressible in a patient, (ii) one or more vectors individually or collectively encoding all essential HSV genes but excluding all cleavage/packaging signals, and (iii) a vhs expression vector encoding a virion host shutoff protein; and then isolating HSV amplicon particles produced by the host cell, the HSV amplicon particles including the transgene. Also disclosed are a system and a kit for preparing HSV amplicon particles, HSV amplicon particles prepared according to the process of the present invention, and their use.

This application claims benefit of U.S. Provisional Application SerialNo. 60/206,497, filed May 23, 2000, which is hereby incorporated byreference in its entirety.

The present invention was made, at least in part, with support from theNational Institutes of Health Grant Nos. R01-NS36420 and R21-DK53160,and AFAR Research Grant. The U.S. government may have certain rights inthis invention.

FIELD OF THE INVENTION

The present invention relates to an improved method for producing herpessimplex virus (“HSV”) amplicons, the resulting HSV amplicons, and theiruse in gene therapy.

BACKGROUND OF THE INVENTION

The ability to deliver genes to the nervous system, and to manipulatetheir expression, may make possible the treatment of numerousneurological disorders. Unfortunately, gene transfer into the centralnervous system (“CNS”) presents several problems including the relativeinaccessibility of the brain and the blood-brain-barrier, and thatneurons of the postnatal brain are post-mitotic. The standard approachfor somatic cell gene transfer, i.e., that of retroviral vectors, is notfeasible for the brain, as retrovirally mediated gene transfer requiresat least one cell division for integration and expression. A number ofnew vectors and non-viral methods have therefore been used for genetransfer in the CNS. Although the first studies of gene transfer in theCNS used an ex vivo approach, i.e., the transplantation ofretrovirally-transduced cells, more recently several groups have alsoused an in vivo approach.

The in vivo approach was initially largely based on the use of theneurotropic herpes simplex virus (“HSV”), however, HSV vectors presentseveral problems, including instability of expression and reversion towild-type.

The genome of HSV-1 is about 150 kb of linear, double-stranded DNA,featuring about 70 genes. Many viral genes may be deleted without thevirus losing its ability to propagate. The “immediately early” (“IE”)genes are transcribed first. They encode trans-acting factors whichregulate expression of other viral genes. The “early” (“E”) geneproducts participate in replication of viral DNA. The late genes encodethe structural components of the virion as well as proteins which turnon transcription of the IE and E genes or disrupt host cell proteintranslation.

After viral entry into the nucleus of a neuron, the viral DNA can entera state of latency, existing as circular episomal elements in thenucleus. While in the latent state, its transcriptional activity isreduced. If the virus does not enter latency, or if it is reactivated,the virus produces numerous infectious particles, which leads rapidly tothe death of the neuron. HSV-1 is efficiently transported betweensynaptically connected neurons, and hence can spread rapidly through thenervous system.

Two types of HSV vectors previously have been utilized for gene transferinto the nervous system. Recombinant HSV vectors involve the removal ofan immediate-early gene within the HSV genome (ICP4, for example), andreplacement with the gene of interest. Although removal of this geneprevents replication and spread of the virus within cells which do notcomplement for the missing HSV protein, all of the other genes withinthe HSV genome are retained. Replication and spread of such viruses invivo is thereby limited, but expression of viral genes within infectedcells continues. Several of the viral expression products may bedirectly toxic to the recipient cell, and expression of viral geneswithin cells expressing MHC antigens can induce harmful immunereactions. In addition, nearly all adults harbor latent herpes simplexviruses within neurons, and the presence of recombinant HSV vectorscould result in recombinations which can produce an actively replicatingwild-type virus. Alternatively, expression of viral genes from therecombinant vector within a cell harboring a latent virus might promotereactivation of the virus. Finally, long-term expression from therecombinant HSV vector in the CNS has not been reliably demonstrated. Itis likely that, except for conditions in which latency is induced, theinability of HSV genomes to integrate within host DNA results insusceptibility to degradation of the vector DNA.

In an attempt to circumvent the difficulties inherent in the recombinantHSV vector, defective HSV vectors were employed as gene transfervehicles within the nervous system. The defective HSV vector is aplasmid-based system, whereby a plasmid vector (termed an amplicon) isgenerated which contains the gene of interest and two cis-acting HSVrecognition signals. These are the origin of DNA replication and thecleavage packaging signal. These sequences encode no HSV gene products.In the presence of HSV proteins provided by a helper virus, the ampliconis replicated and packaged into an HSV coat. This vector thereforeexpresses no viral gene products within the recipient cell, andrecombination with or reactivation of latent viruses by the vector islimited due to the minimal amount of HSV DNA sequence present within thedefective HSV vector genome. The major limitation of this system,however, is the inability to eliminate residual helper virus from thedefective vector stock. The helper virus is often a mutant HSV which,like the recombinant vectors, can only replicate under permissiveconditions in tissue culture. The continued presence of mutant helperHSV within the defective vector stock, however, presents problems whichare similar to those enumerated above in regard to the recombinant HSVvector. This would therefore serve to limit the usefulness of thedefective HSV vector for human applications.

While HSV vectors of reduced toxicity and replication ability have beensuggested, they can still mutate to a more dangerous form, or activate alatent virus, and, since the HSV does not integrate, achieving long-termexpression would be difficult.

To avoid the difficulties raised with the use of helper viruses, newermethods of packaging have been developed that result in “helpervirus-free” amplicon stocks (Fraefel et al., “Helper virus-free transferof herpes simplex virus type 1 plasmid vectors into neural cells,” J.Virol., 70:7190-7197 (1996); Stavropoulos and Strathdee, “An enhancedpackaging system for helper-dependent herpes simplex virus vectors,” J.Virol., 72:7137-43 (1998)). Stocks produced by these means, however, aretypically of low titer (approximately 10⁵ expression units/ml or less),allowing for only modest in vitro experimentation. Such low titersdiscourage investigators from performing the large animal studiesrequired to develop and assess amplicon-directed therapies in mammals,including humans.

The present invention is directed to overcoming these deficiencies inthe art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method forproducing herpes simplex virus (“HSV”) amplicon particles, whichincludes co-transfecting a host cell with the following: (i) an ampliconvector comprising an HSV origin of replication, an HSVcleavage/packaging signal, and a heterologous transgene expressible in apatient, (ii) one or more vectors individually or collectively encodingall essential HSV genes but excluding all cleavage/packaging signals,and (iii) a vhs expression vector encoding a virion host shutoffprotein; and then isolating HSV amplicon particles produced by the hostcell, the HSV amplicon particles including the transgene.

A second aspect of the present invention relates to HSV ampliconparticles produced according to the method of the present invention.

A third aspect of the present invention relates to a system forpreparing HSV amplicon particles which includes: an amplicon vectorcomprising an HSV origin of replication, an HSV cleavage/packagingsignal, and a transgene insertion site; one or more vectors individuallyor collectively encoding all essential HSV genes but excluding allcleavage/packaging signals; and a vhs expression vector encoding avirion host shutoff protein; wherein upon introduction of the systeminto a host cell, the host cell produces herpes simplex virus ampliconparticles.

A fourth aspect of the present invention relates to a kit for preparingHSV amplicon particles which includes: an amplicon vector comprising anHSV origin of replication, an HSV cleavage/packaging signal, and atransgene insertion site; one or more vectors individually orcollectively encoding all essential HSV genes but excluding allcleavage/packaging signals; a vhs expression vector encoding an virionhost shutoff protein; a population of host cells susceptible totransfection by the amplicon vector, the vhs expression vector, and theone or more vectors; and directions for transfecting the host cellsunder conditions to produce HSV amplicon particles.

A fifth aspect of the present invention relates to a method of treatinga neurological disease or disorder which includes providing HSV ampliconparticles of the present invention that include a transgene encoding atherapeutic transgene product and exposing neural or pre-neural cells ofa patient to the HSV amplicon particles under conditions effective forinfective transformation of the neural or pre-neural cells, wherein thetherapeutic transgene product is expressed in vivo in the neural orpre-neural cells, thereby treating the neurological disease or disorder.

A sixth aspect of the present invention relates to a method ofinhibiting development of a neurological disease or disorder whichincludes providing HSV amplicon particles of the present invention thatinclude a transgene encoding a therapeutic transgene product andexposing neural or pre-neural cells of a patient susceptible todevelopment of a neurological disease or disorder to the HSV ampliconparticles under conditions effective for infective transformation of theneural or pre-neural cells of the patient, wherein the therapeutictransgene product is expressed in vivo in the neural or pre-neuralcells, thereby inhibiting development of the neurological disease ordisorder.

A seventh aspect of the present invention relates to a method ofexpressing a therapeutic gene product in a patient which includesproviding HSV amplicon particles of the present invention that include atransgene encoding a therapeutic transgene product and exposing patientcells to the HSV amplicon particles under conditions effective forinfective transformation of the cells, wherein the therapeutic transgeneproduct is expressed in vivo in transformed cells.

In an effort to enhance amplicon titers, the present invention involvesintroduction in trans of a vector including a sequence which encodes avirion host shutoff protein. Co-transfection of this plasmid,specifically one containing the HSV virion host shutoff (“vhs”)protein-encoding gene UL41, with the amplicon and packaging reagentsresults in a 10-fold higher amplicon titer and stocks that do notexhibit the pseudotransduction phenomenon. To further enhance packagingefficiency, the HSV transcriptional activator VP16 was introduced intopackaging cells prior to the packaging components. Pre-loading ofpackaging cells with VP16 led to an additional enhancement of amplicontiters, an effect that did not occur in the absence of vhs. Increasedhelper virus-free amplicon titers resulting from these modificationswill make in vivo transduction experiments more feasible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are maps of suitable amplicon vectors. FIG. 1A is a map ofthe empty amplicon vector pHSVlac, which includes the HSV-1 a segment(cleavage/packaging or pac signal), the HSV-1 c region (origin ofreplication), an ampicillin resistance marker, and an E. coli lacZmarker under control of HSV IE4 promoter and SV40 polyadenylationsignal. FIG. 1B illustrates insertion of a transgene into BamHI siteadjacent the HSV-1 a segment, forming pHSVlac/trans.

FIGS. 2A-B are maps of the HSV-1 genome and the overlapping 5 cosmid setC6Δa48Δa (cos 6Δα, cos 28, cos 14, cos 56, and cos 48Δa) (Fraefel etal., “Helper virus-free transfer of herpes simplex virus type 1 plasmidvectors into neural cells,” J. Virol., 70:7190-7197 (1996), which ishereby incorporated by reference in its entirety). In the HSV-1 genomeof FIG. 2A, only the IE4 gene, ori_(S), and ori_(L) are shown. The asequences, which contain the cleavage/packaging sites, are located atthe junction between long and short segments and at both termini. InFIG. 2B, the deleted a sequences in cos 6Δa and cos 48Δa are indicatedby “X”.

FIG. 3 is a map of the HSV bacterial artificial chromosome (HSV-BAC).

FIG. 4A is a map of pBSKS(vhs), a plasmid vector which includes theHSV-1 vhs coding region (SEQ ID No: 3) operatively coupled to its nativetranscriptional control elements. FIGS. 4B-C show the nucleotidesequence of a 4.3 kb fragment of the HSV-1 genome which contains the vhsgene with its native promoter and polyadenylation signal sequences (SEQID No: 1). The vhs coding sequence is underlined.

FIG. 5 is a map of pGRE₅vp16, a plasmid vector which includes fiveglucocorticoid responsive elements located upstream of a adenovirusmajor late promoter having a TATA box, an HSV vp16 coding sequence (SEQID No: 5), and an SV40 polyadenylation signal. The plasmid also includesan ampicillin resistance marker.

FIGS. 6A-B are graphs which illustrate the effect of vhs expression onhelper virus-free amplicon packaging titers. Theβ-galactosidase-expressing (LacZ) HSV amplicon vector (HSVlac) waspackaged in the absence or presence of pBS(vhs) by either thecosmid-(FIG. 6A) or BAC-based (FIG. 6B) helper virus-free productionstrategy. This pBS(vhs) plasmid possesses the vhs open reading frame aswell as its entire 5′ and 3′ regulatory sequences. Amplicon stocks wereharvested and used to transduce NIH 3T3 cells, and titers weredetermined one day later via enumeration of LacZ-positive cells. Titerdata are expressed as blue-forming units per milliliter (bfu/ml) anderror bars represent standard deviation.

FIGS. 7A-G are images which illustrate the in vitro and in vivo analysisof vhs-mediated enhancement of helper-free amplicon titers. Tenmicroliters of BAC-packaged HSVlac produced without (FIG. 7A) or in thepresence of pBS(vhs) (FIG. 7B) was used to transduce NIH 3T3fibroblasts. LacZ-positive cells were visualized by X-gal histochemistryand images were digitally acquired. Ten microliters of BAC-packagedHSVPrPUC/CMVegfp produced either without (FIG. 7C) or in the presence ofpBS(vhs) (FIG. 7D) was used to transduce NIH 3T3 fibroblasts. Greenfluoresecent protein (GFP)-positive cells were visualized with afluorescent microscope and images digitally acquired. Three microlitersof the same virus samples packaged either in the absence (FIG. 7E) or inthe presence of pBS(vhs) (FIG. 7F) was stereotactically delivered intothe striata of C57BL/6 mice. Animals were sacrificed four days later andprepared for visualization and quantitation of GFP-positive cells.Images used for morphological analyses were digitally acquired at 200×magnification on 40-μm sections. All compartments were processed forcell counting and GFP-positive cell numbers reflect cell countsthroughout the entire injection site (FIG. 7G). The asterisk indicates astatistically significant difference (p<0.001) between amplicon stockspackages with BAC alone and those packaged with BAC in the presence ofpBS(vhs).

FIGS. 8A-D are graphs illustrating the effects of vhs presence duringamplicon packaging on freeze/fracture stability and thermostability.BAC-packaged HSVPrPUC/CMVegfp stocks produced in the presence (circles)or absence (squares) of vhs were incubated at 0° C. (FIG. 8A), 22° C.(FIG. 8B), or 37° C. (FIG. 8C) for varying time periods. At 0, 30, 60,120, and 180 minutes following initiation of the incubations, aliquotswere removed, titered on NIH 3T3 cells, and expression titer datarepresented as green-forming units per milliliter. Another set ofHSVPrPUC/CMVegfp stocks were subjected to a series of freeze-thaw cyclesto determine sensitivity of viral particles to freeze fracture.Following each cycle, aliquots were removed, titered on NIH 3T3 cells,and expression titer data represented as green-forming units permilliliter (gfu/ml; FIG. 8D).

FIGS. 9A-C illustrate the effect of the pre-loading of packaging cellswith VP16 on enhancement of amplicon expression titers only in presenceof vhs. BHK cells were plated and 6 hours later, were transfected with aglucocorticoid-regulated VP16 expression vector (pGRE₅vp16). A subset ofcultures received 100 nM dexamethasone following the VP16 plasmidtransfection. The following day, HSVlac, a β-galactosidase-expressingamplicon, was cosmid-(FIG. 9A) or BAC-packaged (FIG. 9B) in the absenceor presence of the pBS(vhs) plasmid using the modified BHK cultures.Resultant amplicon stocks were titered on NIH 3T3 cells using X-galhistochemistry and titers represented as blue-forming units permilliliter (bfu/ml; FIGS. 9A-B). Error bars represent standarddeviation. Western blot analysis was performed to determine levels ofVP16 expression in various combinations of helper virus-free packagingcomponents (FIG. 9C). Lysates were harvested 48 h following introductionof BAC reagent. Lane designations are the following: BHK cells alone(Lane 1); BHK cells transfected with BAC only (Lane 2); BHKs transfectedwith pGRE₅vp16 24 h prior to BAC transfection in the absence ofdexamethasone (Lane 3); and BHKs transfected with pGRE₅vp16 24 h priorto BAC transfection in the presence of 100 nM dexamethasone (Lane 4).The 65-kDa VP16 protein was detected using a VP16-specific monoclonalantibody and goat anti-mouse secondary antibody in combination with achemiluminescent detection kit.

FIG. 10 is a graph illustrating that the virion-incorporated amplicongenome levels are enhanced by ectopic expression of VP16. BAC-packagedHSVlac stocks prepared in the presence or absence of VP16 and/or vhswere analyzed for levels of genome content using a “real-time”quantitative PCR technique. Nanogram quantities of vector genome wereassayed for each sample and data were expressed as detected amplicongenome per milliliter. Error bars represent standard deviation.

FIG. 11 is a graph illustrating the virion-incorporated amplicon genomelevels are enhanced by ectopic expression of VP16. BAC-packaged HSVlacstocks prepared in the presence or absence of VP16 and/or vhs wereanalyzed for amplicon titer (bfu/ml) using a “real-time” analysis. Errorbars represent standard deviation.

FIG. 12 is a graph illustrating that amplicon stock-mediatedcytotoxicity is not increased by additional expression of vhs and VP16during packaging. BAC-packaged HSVlac stocks prepared in the presence orabsence of VP16 and/or vhs were analyzed on confluent monolayers of NIH3T3 cells for elicited cytotoxicity as determined by an LDHrelease-based assay. Two of the packaging samples that receivedpGRE₅vp16 were also treated with 100 nM dexamethasone 24 hours prior tothe packaging transfection. Equivalent expression units of virus fromeach packaging sample were used in the transductions. Viability datawere represented as normalized cell viability index.

FIG. 13 is a scanning electron micrograph image of purified helper-virusfree HSV-1 amplicon virion stocks prepared using a negative stainingtechnique. Arrows denote individual amplicon particles.

FIG. 14 is an image of a two-dimension gel for polypeptide analysis ofvirion particle stock prepared using helper virus-free procedureaccording to the present invention. Individual spots have been numbered.See Table 2, Example 4, for spot numbering and measurements.

FIG. 15 is an image of a two-dimension gel for polypeptide analysis ofvirion particle stock prepared using helper virus procedure which isknown in the art. Individual spots have been numbered. See Table 2,Example 4, for spot numbering and measurements.

FIGS. 16A-B are difference images of gels shown in FIGS. 14 and 15,showing spots which are increased in FIG. 15 as compared to FIG. 14.FIG. 16B is an enlarged view of the most crowded region. See Table 2,Example 4, for spot numbering and measurements.

FIGS. 17A-C are difference images of gels shown in FIGS. 14 and 15,showing spots which are decreased in FIG. 15 as compared to FIG. 14.FIGS. 17B-C are enlarged views of the two most crowded regions. SeeTable 2, Example 4, for spot numbering and measurements.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method for producingherpes simplex virus (HSV) amplicon particles. This method is carriedout by co-transfecting a host cell with several vectors and thenisolating HSV amplicon particles produced by the host cell. The vectorsused to transfect the host cell include: (i) an amplicon vectorcomprising an HSV origin of replication, an HSV cleavage/packagingsignal, and a heterologous transgene expressible in a patient; (ii) oneor more vectors individually or collectively encoding all essential HSVgenes but excluding all cleavage/packaging signals; and (iii) a vhsexpression vector encoding a virion host shutoff protein. As a result ofthe transgene being included in the HSV amplicon vector, the HSVamplicon particles include the transgene.

The amplicon vector is any HSV amplicon vector which includes an HSVorigin of replication, an HSV cleavage/packaging signal, and aheterologous transgene expressible in a patient. The amplicon vector canalso include a selectable marker gene and an antibiotic resistance gene.

The HSV cleavage/packaging signal can be any suitable cleavage/packagingsignal such that the vector can be packaged into a particle that iscapable of adsorbing to a cell (i.e., which is to be transformed). Asuitable packaging signal is the HSV-1 a segment located atapproximately nucleotides 127-1132 of the a sequence of the HSV-1 virusor its equivalent (Davison et al., “Nucleotide sequences of the jointbetween the L and S segments of herpes simplex virus types 1 and 2,” J.Gen. Virol. 55:315-331 (1981), which is hereby incorporated by referencein its entirety).

The HSV origin of replication can be any suitable origin of replicationwhich allows for replication of the amplicon vector in the host cellwhich is to be used for replication and packaging of the vector into theHSV amplicon particles. A suitable origin of replication is the HSV-1 cregion which contains the HSV-1 ori_(s) segment located at approximatelynucleotides 47-1066 of the HSV-1 virus or its equivalent (McGeogh etal., Nucl. Acids Res. 14:1727-1745 (1986), which is hereby incorporatedby reference in its entirety). Origin of replication signals from otherrelated viruses (e.g., HSV-2) can also be used.

Selectable marker genes are known in the art and include, withoutlimitation, galactokinase, beta-galactosidase, chloramphenicolacetyltransferase, beta-lactamase, green fluorescent protein (“gfp”),alkaline phosphate, etc.

Antibiotic resistance genes are known in the art and include, withoutlimitation, ampicillin, streptomycin, spectromycin, etc.

A number of suitable empty amplicon vectors have previously beendescribed in the art, including without limitation: pHSVlac (ATCCAccession 40544; U.S. Pat. No. 5,501,979 to Geller et al.; Stavropoulosand Strathdee, “An enhanced packaging system for helper-dependent herpessimplex virus vectors,” J. Virol., 72:7137-43 (1998), which are herebyincorporated by reference in their entirety) and pHENK (U.S. Pat. No.6,040,172 to Kaplitt et al., which is hereby incorporated by reference.The pHSVlac vector includes the HSV-1 a segment, the HSV-1 c region, anampicillin resistance marker, and an E. coli lacZ marker. The pHENKvector include the HSV-1 a segment, an HSV-1 ori segment, an ampicillinresistance marker, and an E. coli lacZ marker under control of thepromoter region isolated from the rat preproenkephalin gene (i.e., apromoter operable in brain cells).

These empty amplicon vectors can be modified by introducing therein, atan appropriate restriction site, either a complete transgene which hasalready been assembled or a coding sequence can be ligated into an emptyamplicon vector which already contains appropriate regulatory sequences(promoter, enhancer, polyadenylation signal, transcription terminator,etc.) positioned on either side of the restriction site where the codingsequence is to be inserted, thereby forming the transgene upon ligation.Alternatively, when using the pHSVlac vector, the lacZ coding sequencecan be excised using appropriate restriction enzymes and replaced with acoding sequence for the transgene.

The use of restriction enzymes for cutting DNA and the use of DNA ligaseto ligate together two or more DNA molecules can be performed usingconventional molecular genetic manipulation for subcloning genefragments, as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989); Ausubel et al. (ed.), Current Protocols in Molecular Biology,John Wiley & Sons (New York, N.Y.) (1999 and preceding editions); andU.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which are herebyincorporated by reference in their entirety.

Suitable transgenes will include one or more appropriate promoterelements which are capable of directing the initiation of transcriptionby RNA polymerase, optionally one or more enhancer elements, andsuitable transcription terminators or polyadenylation signals.

Basically, the promoter elements should be selected such that thepromoter will be operable in the cells of the patient which areultimately intended to be transformed (i.e., during gene therapy). Anumber of promoters have been identified which are capable of regulatingexpression within a broad range of cell types. These include, withoutlimitation, HSV immediate-early 4/5 (IE4/5) promoter, cytomegalovirus(“CMV”) promoter, SV40 promoter, and β-actin promoter. Likewise, anumber of other promoters have been identified which are capable ofregulating expression within a narrow range of cell types. Theseinclude, without limitation, neural-specific enolase (NSE) promoter,tyrosine hydroxylase (TH) promoter, GFAP promoter, preproenkephalin(PPE) promoter, myosin heavy chain (MHC) promoter, insulin promoter,cholineacetyltransferase (CHAT) promoter, dopamine β-hydroxylase (DBH)promoter, calmodulin dependent kinase (CamK) promoter, c-fos promoter,c-jun promoter, vascular endothelial growth factor (VEGF) promoter,erythropoietin (EPO) promoter, and EGR-1 promoter.

The transcription termination signal should, likewise, be selected suchthat they will be operable in the cells of the patient which areultimately intended to be transformed. Suitable transcriptiontermination signals include, without limitation, polyA signals of HSVgenes such as the vhs polyadenylation signal, SV40 polyA signal, and CMVIE1 polyA signal.

When used for gene therapy, the transgene encodes a therapeutictransgene product, which can be either a protein or an RNA molecule.

Therapeutic RNA molecules include, without limitation, antisense RNA,inhibitory RNA (RNAi), and an RNA ribozyme. The RNA ribozyme can beeither cis or trans acting, either modifying the RNA transcript of thetransgene to afford a functional RNA molecule or modifying anothernucleic acid molecule. Exemplary RNA molecules include, withoutlimitation, antisense RNA, ribozymes, or RNAi to nucleic acids forhuntingtin, alpha synuclein, scatter factor, amyloid precursor protein,p53, VEGF, etc.

Therapeutic proteins include, without limitation, receptors, signalingmolecules, transcription factors, growth factors, apoptosis inhibitors,apoptosis promoters, DNA replication factors, enzymes, structuralproteins, neural proteins, and histone or non-histone proteins.Exemplary protein receptors include, without limitation, allsteroid/thyroid family members, nerve growth factor (NGF), brain derivedneurotrophic factor (BDNF), neurotrophins 3 and 4/5, glial derivedneurotrophic factor (GDNF), cilary neurotrophic factor (CNTF),persephin, artemin, neurturin, bone morphogenetic factors (BMPs), c-ret,gp130, dopamine receptors (D1-D5), muscarinic and nicotinic cholinergicreceptors, epidermal growth factor (EGF), insulin and insulin-likegrowth factors, leptin, resistin, and orexin. Exemplary proteinsignaling molecules include, without limitation, all of the above-listedreceptors plus MAPKs, ras, rac, ERKs, NFKB, GSK3β, AKT, and PI3KExemplary protein transcription factors include, without limitation,p300, CBP, HIF-1 alpha, NPAS1 and 2, HIF-1β, p53, p73, nurr 1, nurr 77,MASHs, REST, and NCORs. Exemplary neural proteins include, withoutlimitation, neurofilaments, GAP-43, SCG-10, etc. Exemplary enzymesinclude, without limitation, TH, DBH, aromatic aminoacid decarboxylase,parkin, unbiquitin E3 ligases, ubiquitin conjugating enzymes,cholineacetyltransferase, neuropeptide processing enzymes, dopamine,VMAT and other catecholamine transporters. Exemplary histones include,without limitation, H1-5. Exemplary non-histones include, withoutlimitation, ND10 proteins, PML, and HMG proteins. Exemplary pro- andanti-apoptotic proteins include, without limitation, bax, bid, bak,bcl-xs, bcl-xl, bcl-2, caspases, SMACs, and IAPs.

The one or more vectors individually or collectively encoding allessential HSV genes but excluding all cleavage/packaging signals caneither be in the form of a set of vectors or a singlebacterial-artificial chromosome (“BAC”), which is formed, for example,by combining the set of vectors to create a single, double-strandedvector. Preparation and use of a five cosmid set is disclosed in(Fraefel et al., “Helper virus-free transfer of herpes simplex virustype 1 plasmid vectors into neural cells,” J. Virol., 70:7190-7197(1996), which is hereby incorporated by reference in its entirety).Ligation of the cosmids together to form a single BAC is disclosed inStavropoulos and Strathdee, “An enhanced packaging system forhelper-dependent herpes simplex virus vectors,” J. Virol. 72:7137-43(1998), which is hereby incorporated by reference in its entirety). TheBAC described in Stavropoulos and Strathdee includes a pac cassetteinserted at a BamHI site located within the UL41 coding sequence,thereby disrupting expression of the HSV-1 virion host shutoff protein.

By “essential HSV genes”, it is intended that the one or more vectorsinclude all genes which encode polypeptides that are necessary forreplication of the amplicon vector and structural assembly of theamplicon particles. Thus, in the absence of such genes, the ampliconvector is not properly replicated and packaged within a capsid to forman amplicon particle capable of adsorption. Such “essential HSV genes”have previously been reported in review articles by Roizman (“TheFunction of Herpes Simplex Virus Genes: A Primer for Genetic Engineeringof Novel Vectors,” Proc. Natl. Acad. Sci. USA 93:11307-11312 (1996);“HSV Gene Functions: What Have We Learned That Could Be GenerallyApplication to its Near and Distant Cousins?” Acta Virologica43(2-3):75-80 (1999), which are hereby incorporated by reference intheir entirety. Another source for identifying such essential genes isavailable at the Internet site operated by the Los Alamos NationalLaboratory, Bioscience Division, which reports the entire HSV-1 genomeand includes a table identifying the essential HSV-1 genes. The genescurrently identified as essential are listed in Table 1 below.

TABLE 1 Essential HSV-1 Genes Genbank Gene* Protein (Function) I.D. No.Accession No.** UL1 virion glycoprotein L (gL) 136775 CAA32337 UL5component of DNA helicase-primase complex 74000 CAA32341 UL6 minorcapsid protein 136794 CAA32342 UL7 unknown 136798 CAA32343 UL8 DNAhelicase/primase complex associated protein 136802 CAA32344 UL8.5unknown*** — — UL9 oil-binding protein 136806 CAA32345 UL15 DNAcleavage/packaging protein 139646 CAA32330 UL17 tegument protein 136835CAA32329 UL18 capsid protein, VP23 139191 CAA32331 UL19 major capsidprotein,VP5 137571 CAA32332 UL22 virion glycoprotein H, gH 138315CAA32335 UL25 DNA packaging virion protein 136863 CAA32317 UL26 serineprotease, self-cleaves to form VP21 & VP24 139233 CAA32318 UL26.5 capsidscaffolding protein, VP22a 1944539 CAA32319 UL27 virion glycoprotein B,gB 138194 CAA32320 UL28 DNA cleavage and packaging protein, ICP18.5124088 CAA32321 UL29 single-stranded DNA binding protein, ICP8 118746CAA32322 UL30 DNA polymerase 118878 CAA32323 UL31 UL34-associatednuclear protein 136875 CAA32324 UL32 cleavage and packaging protein136879 CAA32307 UL33 capsid packaging protein 136883 CAA32308 UL34membrane-associated virion protein 136888 CAA32309 UL36 very largetegument protein, ICP1/2 135576 CAA32311 UL37 tegument protein, ICP32136894 CAA32312 UL38 capsid protein, VP19C 418280 CAA32313 UL42 DNApolymerase accessory protein 136905 CAA32305 UL48 alpha trans-inducingfactor, VP16 114359 CAA32298 UL49 putative microtubule-associatedprotein, VP22 136927 CAA32299 UL49.5 membrane-associated virion protein1944541 CAA32300 UL52 component of DNA helicase/primase complex 136939CAA32288 UL54 regulation and transportation of RNA, ICP27 124180CAA32290 α4 (RS1) positive and negative gene regulator, ICP4 124141CAA32286 CAA32278 US6 virion glycoprotein D, gD 73741 CAA32283 *Thecomplete genome of HSV-1 is reported at Genbank Accession No. X14112,which is hereby incorporated by reference in its entirety. **Each of thelisted Accession Nos. which report an amino acid sequence for theencoded proteins is hereby incorporated by reference in its entirety.***UL8.5 maps to a transcript which overlaps and is in frame with thecarboxyl terminal of UL9 (Baradaran et al., “Transcriptional analysis ofthe region of the herpes simplex virus type 1 genomecontaining the UL8,UL9, and UL10 genes and identification of a novel delayed-early geneproduct, OBPC,” J. Virol. 68 (7):4251–4261 (1994), which is herebyincorporated by reference in its entirety).

The vhs vector can encode a virion host shutoff (“vhs”) protein which iseffective in regulating host cell transcription and translationactivities. The vhs vector includes a DNA molecule encoding a vhsprotein, which DNA molecule is operably coupled 5′ to a promoter whichis functional in the host cell and 3′ to a transcription terminatorwhich also is functional in the host cell.

One suitable vhs protein is the human herpesvirus 1 vhs protein, whichhas an amino acid sequence according to SEQ ID No: 2 as follows:

Met Gly Leu Phe Gly Met Met Lys Phe Ala His Thr His His Leu Val  1               5                  10                  15Lys Arg Arg Gly Leu Gly Ala Pro Ala Gly Tyr Phe Thr Pro Ile Ala             20                  25                  30Val Asp Leu Trp Asn Val Met Tyr Thr Leu Val Val Lys Tyr Gln Arg         35                  40                  45Arg Tyr Pro Ser Tyr Asp Arg Glu Ala Ile Thr Leu His Cys Leu Cys     50                  55                  60Arg Leu Leu Lys Val Phe Thr Gln Lys Ser Leu Phe Pro Ile Phe Val 65                  70                  75                  80Thr Asp Arg Gly Val Asn Cys Met Glu Pro Val Val Phe Gly Ala Lys                 85                  90                  95Ala Ile Leu Ala Arg Thr Thr Ala Gln Cys Arg Thr Asp Glu Glu Ala            100                 105                 110Ser Asp Val Asp Ala Ser Pro Pro Pro Ser Pro Ile Thr Asp Ser Arg        115                 120                 125Pro Ser Ser Ala Phe Ser Asn Met Arg Arg Arg Gly Thr Ser Leu Ala    130                 135                 140Ser Gly Thr Arg Gly Thr Ala Gly Ser Gly Ala Ala Leu Pro Ser Ala145                 150                 155                 160Ala Pro Ser Lys Pro Ala Leu Arg Leu Ala His Leu Phe Cys Ile Arg                165                 170                 175Val Leu Arg Ala Leu Gly Tyr Ala Tyr Ile Asn Ser Gly Gln Leu Glu            180                 185                 190Ala Asp Asp Ala Cys Ala Asn Leu Tyr His Thr Asn Thr Val Ala Tyr        195                 200                 205Val Tyr Thr Thr Asp Thr Asp Leu Leu Leu Met Gly Cys Asp Ile Val    210                 215                 220Leu Asp Ile Ser Ala Cys Tyr Ile Pro Thr Ile Asn Cys Arg Asp Ile225                 230                 235                 240Leu Lys Tyr Phe Lys Met Ser Tyr Pro Gln Phe Leu Ala Leu Phe Val                245                 250                 255Arg Cys His Thr Asp Leu His Pro Asn Asn Thr Tyr Ala Ser Val Glu            260                 265                 270Asp Val Leu Arg Glu Cys His Trp Thr Pro Pro Ser Arg Ser Gln Thr        275                 280                 285Arg Arg Ala Ile Arg Arg Glu His Thr Ser Ser Arg Ser Thr Glu Thr    290                 295                 300Arg Pro Pro Leu Pro Pro Ala Ala Gly Gly Thr Glu Thr Arg Val Ser305                 310                 315                 320Trp Thr Glu Ile Leu Thr Gln Gln Ile Ala Gly Gly Tyr Glu Asp Asp                325                 330                 335Glu Asp Leu Pro Leu Asp Pro Arg Asp Val Thr Gly Gly His Pro Gly            340                 345                 350Pro Arg Ser Ser Ser Ser Glu Ile Leu Thr Pro Pro Glu Leu Val Gln        355                 360                 365Val Pro Asn Ala Gln Leu Leu Glu Glu His Arg Ser Tyr Val Ala Asn    370                 375                 380Pro Arg Arg His Val Ile His Asp Ala Pro Glu Ser Leu Asp Trp Leu385                 390                 395                 400Pro Asp Pro Met Thr Ile Thr Glu Leu Val Glu His Arg Tyr Ile Lys                405                 410                 415Tyr Val Ile Ser Leu Ile Gly Pro Lys Glu Arg Gly Pro Trp Thr Leu            420                 425                 430Leu Lys Arg Leu Pro Ile Tyr Gln Asp Ile Arg Asp Glu Asn Leu Ala        435                 440                 445Arg Ser Ile Val Thr Arg His Ile Thr Ala Pro Asp Ile Ala Asp Arg    450                 455                 460Phe Leu Glu Gln Leu Arg Thr Gln Ala Pro Pro Pro Ala Phe Tyr Lys465                 470                 475                 480Asp Val Leu Ala Lys Phe Trp Asp Glu                 485This protein is encoded by a DNA molecule having a nucleotide sequenceaccording to SEQ ID No: 3 as follows:

atgggtttgt tcgggatgat gaagtttgcc cacacacacc atctggtcaa gcgccggggc   60cttggggccc cggccgggta cttcaccccc attgccgtgg acctgtggaa cgtcatgtac  120acgttggtgg tcaaatatca gcgccgatac cccagttacg accgcgaggc cattacgcta  180cactgcctct gtcgcttatt aaaggtgttt acccaaaagt cccttttccc catcttcgtt  240accgatcgcg gggtcaattg tatggagccg gttgtgtttg gagccaaggc catcctggcc  300cgcacgacgg cccagtgccg gacggacgag gaggccagtg acgtggacgc ctctccaccg  360ccttccccca tcaccgactc cagacccagc tctgcctttt ccaacatgcg ccggcgcggc  420acctctctgg cctcggggac ccgggggacg gccgggtccg gagccgcgct gccgtccgcc  480gcgccctcga agccggccct gcgtctggcg catctgttct gtattcgcgt tctccgggcc  540ctggggtacg cctacattaa ctcgggtcag ctggaggcgg acgatgcctg cgccaacctc  600tatcacacca acacggtcgc gtacgtgtac accacggaca ctgacctcct gttgatgggc  660tgtgatattg tgttggatat tagcgcctgc tacattccca cgatcaactg tcgcgatata  720ctaaagtact ttaagatgag ctacccccag ttcctggcct ctttgtccgc tgccacaccg  780acctccatcc caataacacc tacgcctccg tggaggatgt gctgcgcgaa tgtcactgga  840cccccccgag tcgctctcag acccggcggg ccatccgccg ggaacacacc agctcgcgct  900ccacggaaac caggccccct ctgccgccgg ccgccggcgg caccgagacg cgcgtctcgt  960ggaccgaaat tctaacccaa cagatcgccg gcggatacga agacgacgag gacctccccc 1020tggatccccg ggacgttacc gggggccacc ccggccccag gtcgtcctcc tcggagatac 1080tcaccccgcc cgagctcgtc caggtcccga acgcgcagct gctggaagag caccgcagtt 1140atgtggccaa cccgcgacgc cacgtcatcc acgacgcccc agagtccctg gactggctcc 1200ccgatcccat gaccatcacc gagctggtgg aacaccgcta cattaagtac gtcatatcgc 1260ttatcggccc caaggagcgg gggccgtgga ctcttctgaa acgcctgcct atctaccagg 1320acatccgcga cgaaaacctg gcgcgatcta tcgtgacccg gcatatcacg gcccctgata 1380tcgccgacag gtttctggag cagttgcgga cccaggcccc cccacccgcg ttctacaagg 1440acgtcctggc caaattctgg gacgagtag 1469The amino acid and encoding nucleotide sequences of human HSV-1 vhs arereported at Genbank Accession Nos. CAA96525 and Z72338, which are herebyincorporated by reference in their entirety. The above-listed nucleotidesequence corresponds to nt 1287-2756 of SEQ ID No: 1.

Other suitable vhs proteins include human herpesvirus 2 vhs protein,whose amino acid and encoding nucleotide sequences are reported,respectively, as Genbank Accession Nos. AAC58447 and AF007816, which arehereby incorporated by reference in their entirety; human herpesvirus 3vhs protein, whose amino acid and sequence is reported as GenbankAccession No. P09275, which is hereby incorporated by reference in itsentirety; bovine herpesvirus 1 vhs protein, whose amino acid andencoding nucleotide sequences are reported, respectively, as GenbankAccession Nos. CAA90927 and Z54206, which are hereby incorporated byreference in their entirety; bovine herpesvirus 1.1 vhs protein, whoseamino acid and encoding nucleotide sequences are reported, respectively,as Genbank Accession Nos.

NP_(—)045317 and NC_(—)001847, which are hereby incorporated byreference in their entirety; gallid herpesvirus 1 vhs protein, whoseamino acid and encoding nucleotide sequences are reported, respectively,as Genbank Accession Nos. AAD56213 and AF168792, which are herebyincorporated by reference in their entirety; gallid herpesvirus 2 vhsprotein, whose amino acid and encoding nucleotide sequences arereported, respectively, as Genbank Accession Nos. AAA80558 and L40429,which are hereby incorporated by reference in their entirety; suidherpesvirus 1 vhs protein, whose amino acid and sequence is reported asGenbank Accession No. P36314, which is hereby incorporated by referencein its entirety; baboon herpesvirus 2 vhs protein, whose amino acid andencoding nucleotide sequences are reported, respectively, as GenbankAccession Nos. AAG01880 and AF294581, which are hereby incorporated byreference in their entirety; pseudorabies virus vhs protein, whose aminoacid and encoding nucleotide sequences are reported, respectively, asGenbank Accession Nos. AAB25948 and S57917, which are herebyincorporated by reference in their entirety; cercopithecine herpesvirus7 vhs protein, whose amino acid and encoding nucleotide sequences arereported, respectively, as Genbank Accession Nos. NP_(—)077432 andNC_(—)002686, which are hereby incorporated by reference in theirentirety; meleagrid herpesvirus 1 vhs protein, whose amino acid andencoding nucleotide sequences are reported, respectively, as GenbankAccession Nos. Np_(—)073335 and NC_(—)002641, which are herebyincorporated by reference in their entirety; equine herpesvirus 1 vhsprotein, whose amino acid and encoding nucleotide sequences arereported, respectively, as Genbank Accession Nos. NP_(—)041028 andNC_(—)001491, which are hereby incorporated by reference in theirentirety; and equine herpesvirus 4 vhs protein, whose amino acidsequence is reported as Genbank Accession No. T42562, which is herebyincorporated by reference in its entirety.

According to one approach, the vhs vector includes a DNA moleculeencoding the HSV virion host shutoff protein operatively coupled to itsnative transcriptional control elements. A vector of this type isprepared by excising an approximately 4.3 kb HpaI/HindIII restrictionfragment from the previously reported cosmid56 (Cunningham and Davison,“A cosmid-based system for construction mutants of herpes simplex type1,” Virology, 197:116-124 (1993), which is hereby incorporated byreference in its entirety) and cloning the fragment into pBSKSII(Stratagene, Inc.) to create pBSKS(vhs). A map of pBSKS(vhs) isillustrated in FIG. 4A. The 4.3 kb fragment includes nts 89658-93923(complement) of the HSV-1 genome (SEQ ID No: 1, see FIGS. 4B-C), asreported at Genbank Accession No. X14112, which is hereby incorporatedby reference in its entirety.

Optionally, the host cell which is co-transfected also expresses asuitable VP16 tegument protein. This can be achieved either by (a)transfecting the host cell prior to the co-transfection step with avector encoding the VP16 protein, or (b) co-transfecting a host cellwhich stably expresses the VP16 protein.

One suitable VP16 protein is the HSV-1 VP16 protein, which ischaracterized by an amino acid sequence according to SEQ ID No: 4 asfollows:

Met Asp Leu Leu Val Asp Glu Leu Phe Ala Asp Met Asn Ala Asp Gly  1               5                  10                  15Ala Ser Pro Pro Pro Pro Arg Pro Ala Gly Gly Pro Lys Asn Thr Pro             20                  25                  30Ala Ala Pro Pro Leu Tyr Ala Thr Gly Arg Leu Ser Gln Ala Gln Leu         35                  40                  45Met Pro Ser Pro Pro Met Pro Val Pro Pro Ala Ala Leu Phe Asn Arg     50                  55                  60Leu Leu Asp Asp Leu Gly Phe Ser Ala Gly Pro Ala Leu Cys Thr Met 65                  70                  75                  80Leu Asp Thr Trp Asn Glu Asp Leu Phe Ser Ala Leu Pro Thr Asn Ala                 85                  90                  95Asp Leu Tyr Arg Glu Cys Lys Phe Leu Ser Thr Leu Pro Ser Asp Val            100                 105                 110Val Glu Trp Gly Asp Ala Tyr Val Pro Glu Arg Thr Gln Ile Asp Ile        115                 120                 125Arg Ala His Gly Asp Val Ala Phe Pro Thr Leu Pro Ala Thr Arg Asp    130                 135                 140Gly Leu Gly Leu Tyr Tyr Glu Ala Leu Ser Arg Phe Phe His Ala Glu145                 150                 155                 160Leu Arg Ala Arg Glu Glu Ser Tyr Arg Thr Val Leu Ala Asn Phe Cys                165                 170                 175Ser Ala Leu Tyr Arg Tyr Leu Arg Ala Ser Val Arg Gln Leu His Arg            180                 185                 190Gln Ala His Met Arg Gly Arg Asp Arg Asp Leu Gly Glu Met Leu Arg        195                 200                 205Ala Thr Ile Ala Asp Arg Tyr Tyr Arg Glu Thr Ala Arg Leu Ala Arg    210                 215                 220Val Leu Phe Leu His Leu Tyr Leu Phe Leu Thr Arg Glu Ile Leu Trp225                 230                 235                 240Ala Ala Tyr Ala Glu Gln Met Met Arg Pro Asp Leu Phe Asp Cys Leu                245                 250                 255Cys Cys Asp Leu Glu Ser Trp Arg Gln Leu Ala Gly Leu Phe Gln Pro            260                 265                 270Phe Met Phe Val Asn Gly Ala Leu Thr Val Arg Gly Val Pro Ile Glu        275                 280                 285Ala Arg Arg Leu Arg Glu Leu Asn His Ile Arg Glu His Leu Asn Leu    290                 295                 300Pro Leu Val Arg Ser Ala Ala Thr Glu Glu Pro Gly Ala Pro Leu Thr305                 310                 315                 320Thr Pro Pro Thr Leu His Gly Asn Gln Ala Arg Ala Ser Gly Tyr Phe                325                 330                 335Met Val Leu Ile Arg Ala Lys Leu Asp Ser Tyr Ser Ser Phe Thr Thr            340                 345                 350Ser Pro Ser Glu Ala Val Met Arg Glu His Ala Tyr Ser Arg Ala Arg        355                 360                 365Thr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly Leu Leu Asp Leu Pro    370                 375                 380Asp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala Ala Pro Arg Leu Ser385                 390                 395                 400Phe Leu Pro Ala Gly His Thr Arg Arg Leu Ser Thr Ala Pro Pro Thr                405                 410                 415Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala            420                 425                 430Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly        435                 440                 445Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro    450                 455                 460Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr465                 470                 475                 480Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly                485                 490The DNA molecule encoding HSV-1 vp16 has a nucleotide sequence accordingto SEQ ID No: 5 as follows:

atggacctct tggtcgacga gctgtttgcc gacatgaacg cggacggcgc ttcgccaccg   60cccccccgcc cggccggggg tcccaaaaac accccggcgg cccccccgct gtacgcaacg  120gggcgcctga gccaggccca gctcatgccc tccccaccca tgcccgtccc ccccgccgcc  180ctctttaacc gtctcctcga cgacttgggc tttagcgcgg gccccgcgct atgtaccatg  240ctcgatacct ggaacgagga tctgttttcg gcgctaccga ccaacgccga cctgtaccgg  300gagtgtaaat tcctatcaac gctgcccagc gatgtggtgg aatgggggga cgcgtacgtc  360cccgaacgca cccaaatcga cattcgcgcc cacggcgacg tggccttccc tacgcttccg  420gccacccgcg acggcctcgg gctctactac gaagcgctct ctcgtttctt ccacgccgag  480ctacgggcgc gggaggagag ctatcgaacc gtgttggcca acttctgctc ggccctgtac  540cggtacctgc gcgccagcgt ccggcagctg caccgccagg cgcacatgcg cggacgcgat  600cgcgacctgg gagaaatgct gcgcgccacg atcgcggaca ggtactaccg agagaccgct  660cgtctggcgc gtgttttgtt tttgcatttg tatctatttt tgacccgcga gatcctatgg  720gccgcgtacg ccgagcagat gatgcggccc gacctgtttg actgcctctg ttgcgacctg  780gagagctggc gtcagttggc gggtctgttc cagcccttca tgttcgtcaa cggagcgctc  840accgtccggg gagtgccaat cgaggcccgc cggctgcggg agctaaacca cattcgcgag  900caccttaacc tcccgctggt gcgcagcgcg gctacggagg agccaggggc gccgttgacg  960acccctccca ccctgcatgg caaccaggcc cgcgcctctg ggtactttat ggtgttgatt 1020cgggcgaagt tggactcgta ttccagcttc acgacctcgc cctccgaggc ggtcatgcgg 1080gaacacgcgt acagccgcgc gcgtacgaaa aacaattacg ggtctaccat cgagggcctg 1140ctcgatctcc cggacgacga cgcccccgaa gaggcggggc tggcggctcc gcgcctgtcc 1200tttctccccg cgggacacac gcgcagactg tcgacggccc ccccgaccga tgtcagcctg 1260ggggacgagc tccacttaga cggcgaggac gtggcgatgg cgcatgccga cgcgctagac 1320gatttcgatc tggacatgtt gggggacggg gattccccgg ggccgggatt taccccccac 1380gactccgccc cctacggcgc tctggatatg gccgacttcg agtttgagca gatgtttacc 1440gatgcccttg gaattgacga gtacggtggg tag 1473The amino acid and encoding nucleotide sequence of human HSV-1 VP16 arereported, respectively, as Genbank Accession Nos. CAA32304 and X14112,which are hereby incorporated by reference in their entirety.

Other suitable VP16 proteins include human herpesvirus 2 VP16 protein,whose amino acid and encoding nucleotide sequences are reported,respectively, as Genbank Accession Nos. NP_(—)044518 and NC_(—)001798,which are hereby incorporated by reference in their entirety; bovineherpesvirus 1 VP16 protein, whose amino acid and encoding nucleotidesequences are reported, respectively, as Genbank Accession Nos. CAA90922and Z54206, which are hereby incorporated by reference in theirentirety; bovine herpesvirus 1.1 VP16 protein, whose amino acid andencoding nucleotide sequences are reported, respectively, as GenbankAccession Nos. NP_(—)045311 and NC_(—)001847, which are herebyincorporated by reference in their entirety; gallid herpesvirus 1 VP16protein, whose amino acid and encoding nucleotide sequences arereported, respectively, as Genbank Accession Nos. BAA32584 and AB012572,which are hereby incorporated by reference in their entirety; gallidherpesvirus 2 VP16 protein, whose amino acid and encoding nucleotidesequences are reported, respectively, as Genbank Accession Nos.NP_(—)057810 and NC_(—)002229, which are hereby incorporated byreference in their entirety; meleagrid herpesvirus 1 VP16 protein, whoseamino acid and encoding nucleotide sequences are reported, respectively,as Genbank Accession Nos. AAG30088 and AF282130, which are herebyincorporated by reference in their entirety; and equine herpesvirus 4VP16 protein, whose amino acid and encoding nucleotide sequences arereported as Genbank Accession Nos. NP_(—)045229 and NC_(—)001844, whichare hereby incorporated by reference in their entirety.

When performing an initial transfection step prior to co-transfection,the transfection with a vector encoding the VP16 protein can be carriedout at least about 1 hour before the co-transfection step, morepreferably at least about 4 hours before, and most preferably at leastabout 12 hours before. Maximal amplicon particle titers have beenachieved following transfection of host cells (with VP16) about 24 hoursprior to the co-transfection step described below. When priortransfection of the host cell is carried out, a preferred vectorencoding the HSV-1 VP16 protein is vector pGRE₅vp16, whose structure isillustrated in FIG. 5.

In host cells transiently expressing VP16, the plasmid encoding VP16 islost in up to about 50% of the cells per doubling of the cellpopulation.

Stable expression of VP16 can be achieved either using a stable plasmidwhich is copied and partitioned among dividing host cells withacceptable fidelity or by integration of the VP16 into the host cellgenome. Plasmids which are stable in vitro cell lines are known in theart and can be used to introduce UL48 thereon. Also, integration can becarried out according to known procedures.

Preparation of HSV amplicon particles can be carried out byco-transfecting a suitable host cell with (i) the amplicon vector, (ii)either the set of cosmid vectors or BAC, and (iii) the vhs expressionvector. Basically, the various vectors are introduced into a singlemedium (e.g., Opti-MEM available from Gibco-BRL, Bethesda, Md.) within acontainer (e.g., sterile polypropylene tube), forming a DNA mix. Theweight ratio of BAC:amplicon vector is between about 1-10:1, preferablyabout 5-10:1, and the weight ratio of 5 cosmid set (in total):ampliconvector is between about 1-10:1, preferably about 2-7:1. The DNA mix islater introduced into a container (with Lipofectanine reagent) which hasbeen seeded with the host cells to be co-transfected. Thereafter, thetransfection mix is diluted with an equal volume of a selection medium(e.g., DMEM plus 20% FBS, 2% penicillin/streptomycin, and 2 mMhexamethylene bis-acetamide (HMBA)) and incubated for several days.Virion particles are released from the host cells by sonication andpurified from host cell protein/membrane components viaultracentrifugation.

When prior transfection is effected, allowing the host cells to expressHSV-1 VP16 prior to co-transfection as described above, the cells platedfor packaging were first allowed to adhere to a culture dish andsubsequently transfected with pGRE₅vp16 using Lipofectamine reagent.Following suitable incubation, the transfection mix was removed,complete medium (e.g., DMEM plus 10% FBS, 1% penicillin/streptomycin)was added, and the cultures were incubated at 37° C. until the packagingco-transfection step described above.

Suitable host cells which can be co-transfected for preparation of HSVamplicon particles are eukaryotic cells, preferably mammalian cells.Exemplary host cells include, without limitation, BHK cells, NIH 3T3cells, 2-2 cells, 293 cells, and RR1 cells.

When the HSV amplicon particles are harvested from the host cell medium,the amplicon particles are substantially pure (i.e., free of any othervirion particles) and present at a concentration of greater than about1×10⁶ particles per milliliter. To further enhance the use of theamplicon particles, the resulting stock can also be concentrated, whichaffords a stock of isolated HSV amplicon particles at a concentration ofat least about 1×10⁷ particles per milliliter.

The resulting amplicon particles produced according to the presentinvention, i.e., in the presence of vhs and, optionally VP16, both ofwhich can be expressed in host cells prior to packaging, aresubstantially different in kind from the virion particles which can beprepared using known helper virus methods (see Examples 1 and 4).

The concentrated stock of HSV amplicon particles is effectively acomposition of the HSV amplicon particles in a suitable carrier.Alternatively, the HSV amplicon particles may also be administered ininjectable dosages by dissolution or suspension of these materials in aphysiologically acceptable diluent with a pharmaceutical carrier. Suchcarriers include sterile liquids, such as water and oils, with orwithout the addition of a surfactant and other pharmaceutically andphysiologically acceptable carriers, including adjuvants, excipients orstabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols, such as propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions.

For use as aerosols, the HSV amplicon particles, in solution orsuspension, may be packaged in a pressurized aerosol container togetherwith suitable propellants, for example, hydrocarbon propellants likepropane, butane, or isobutane with conventional adjuvants. The materialsof the present invention also may be administered in a non-pressurizedform such as in a nebulizer or atomizer.

The pharmaceutical composition is preferably in liquid form, such as asolution, suspension, or emulsion. Typically, the composition willcontain at least about 1×10⁷ amplicon particles/ml, together with thecarrier, excipient, stabilizer, etc.

A further aspect of the present invention relates to a system forpreparing HSV amplicon particles. The system includes (i) an emptyamplicon vector as described above, which includes an HSV origin ofreplication, an HSV cleavage/packaging signal, and a transgene insertionsite (at which a transgene may be inserted, as described above), (ii)one or more vectors individually or collectively encoding all essentialHSV genes but excluding all cleavage/packaging signals, and (iii) a vhsexpression vector encoding a virion host shutoff protein. The vhsexpression vector is of the type described above. The system ischaracterized as being able to produce HSV amplicon particles of thepresent invention when the system is introduced (i.e., co-transfected)into a suitable host cell. The system may further include, as describedabove, a host cell which stably expresses an HSV VP16 protein and/or avector encoding the HSV VP16 protein.

Yet another aspect of the present invention relates to a kit forpreparing HSV amplicon particles of the present invention. The kitsincludes: (i) an amplicon vector comprising an HSV origin ofreplication, an HSV cleavage/packaging signal, and a transgene insertionsite (at which a transgene may be inserted, as described above), (ii)one or more vectors individually or collectively encoding all essentialHSV genes but excluding all cleavage/packaging signals, (iii) a vhsexpression vector encoding an virion host shutoff protein, (iv) apopulation of host cells susceptible to transfection by the ampliconvector, the vhs expression vector, and the one or more vectors, and (v)directions for transfecting the host cells under conditions to produceHSV amplicon particles. The vhs expression vector is of the typedescribed above. The kit may further include, as described above, a hostcell which stably expresses an HSV VP16 protein and/or a vector encodingthe HSV VP16 protein.

Yet another aspect of the present invention relates generally to amethod of expressing a therapeutic gene product in a patient using theHSV amplicon particles of the present invention which contain atransgene encoding a therapeutic gene product. Basically, this method iscarried out by providing such HSV amplicon particles and exposingpatient cells to the HSV amplicon particles under conditions effectivefor infective transformation of the cells, wherein the therapeutictransgene product is expressed in vivo in transformed cells. As notedbelow, transformation of the patient cells can be carried out in vivo orex vivo.

HSV-1 has a wide host range and infects many cell types in mammals andbirds (including chickens, rats, mice, monkeys, humans) (Spear et al.,DNA Tumor Viruses, pp. 615-746, Tooze, ed., Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1981), which is herebyincorporated by reference in its entirety). HSV-1 can lytically infect awide variety of cells including, e.g., neurons, fibroblasts, andmacrophages. In addition, HSV-1 infects post-mitotic neurons in adultanimals and can be maintained indefinitely in a latent state (Stevens,Curr. Topics in Microbiol. and Immunol. 70:31-50 (1975), which is herebyincorporated by reference in its entirety). Two lines of evidencesuggest that HSV-1 can infect most, if not all, kinds of neurons in thecentral nervous system. First, following inoculation of HSV-1 in theperiphery, a burst of virus production ascends the neuroaxis, initiallyin the sensory or motor neurons innervating the site of inoculation,then in the spinal cord, brain stem, cerebellum, and cerebral cortex(Koprowski, In Persistent Viruses, pp. 691-699, Stevens, ed., AcademicPress, New York, N.Y. (1978), which is hereby incorporated by referencein its entirety). Second, attempts to mimic HSV-1 latency in tissueculture with different preparations of neurons have required hightemperature, DNA synthesis inhibitors, and antisera directed againstHSV-1 virions to prevent lytic infection for spreading to all neurons(Wigdahl et al., Proc. Natl. Acad. Sci. USA 81:6217-6201 (1984), whichis hereby incorporated by reference in its entirety).

Because HSV-1 infects a wide range of animals, the HSV ampliconparticles of the present invention can be used on a wide variety ofmammals and birds. Preferably, the HSV amplicon particles are used onmammals, most preferably humans, to effect expression of the therapeutictransgene product. Thus, as used herein, patient refers generally tomammals and birds, as well as humans specifically.

When exposing the patient cells to the HSV amplicon particles, an invivo route of delivery is performed by administering the HSV ampliconparticles directly to the patient cells which are to be transformed. Theadministering can be achieved in a manner which is suitable to effectdelivery and subsequent patient cell transformation, including, withoutlimitation, intraparenchymal, intramuscular, intravenous,intracerebroventricular, subcutaneous, or intramucosal delivery.

Alternatively, an ex vivo route of delivery is performed by providingpatient cells (either removed from the patient or obtained from adonor), exposing the cells ex vivo to the HSV amplicon particles, andthen introducing the transformed cells into the patient. Stem cells,embryonic or progenitor, can be effectively transformed and thenintroduced into the patient at a desired location. For non-motiletransformed cells, such cells are preferably administered to the patientat the site where the cells are intended to reside. For actively orpassively motile transformed cells, such cells may be administered in amanner which is effective to deliver the transformed cells into thepatient. Suitable delivery routes include, without limitation,intraparenchymal, intramuscular, intravenous, intracerebroventricular,subcutaneous, or intramucosal delivery.

Still another aspect of the present invention relates to a method oftreating a neurological disease or disorder using the HSV ampliconparticles of the present invention which include a transgene encoding atherapeutic transgene product. Basically, this method is carried out byproviding such HSV amplicon particles and exposing patient neural orpre-neural cells to the HSV amplicon particles under conditionseffective for infective transformation of neural or pre-neural cells ofthe patient, wherein the therapeutic tansgene product is expressed invivo by the neural or pre-neural cells, thereby treating theneurological disease or disorder.

As noted above, transformation can be effected either in vivo or ex vivo(i.e., using differentiated neural cells, neural stem cells, orembryonic stem cells which differentiate into neural cells). A preferredin vivo route of delivery is administering the HSV amplicon particlesdirectly to neural cells which are to be treated using, e.g., thedelivery routes listed above.

Neuronal diseases or disorders which can be treated include lysosomalstorage diseases (e.g., by expressing MPS 1-VIII, hexoaminidase A/B,etc.), Lesch-Nyhan syndrome (e.g., by expressing HPRT), amyloidpolyneuropathy (e.g., by expressing β-amyloid converting enzyme (BACE)or amyloid antisense), Alzheimer's Disease (e.g., by expressing NGF,CHAT, BACE, etc.), retinoblastoma (e.g., by expressing pRB), Duchenne'smuscular dystrophy (e.g., by expressing Dystrophin), Parkinson's Disease(e.g., by expressing GDNF, Bcl-2, TH, AADC, VMAT, antisense to mutantalpha-synuclein, etc.), Diffuse Lewy Body disease (e.g., by expressingheat shock proteins, parkin, or antisense or RNAi to alpha-synuclein),stroke (e.g., by expressing Bcl-2, HIF-DN, BMP7, GDNF, other growthfactors), brain tumor (e.g., by expressing angiostatin, antisense VEGF,antisense or ribozyme to EGF or scatter factor, pro-apoptotic proteins),epilepsy (e.g., by expressing GAD65, GAD67, pro-apoptotic proteins intofocus), or arteriovascular malformation (e.g., by expressingproapoptotic proteins).

Likewise, the HSV amplicon particles of the present invention whichinclude a transgene encoding a therapeutic transgene product can also beused according to a method of inhibiting development of a neurologicaldisease or disorder. Basically, this method is carried out by providingsuch HSV amplicon particles and exposing neural or pre-neural cells ofthe patient who is susceptible to development of a neurological diseaseor disorder to the HSV amplicon particles under conditions effective forinfective transformation of the neural or pre-neural cells, wherein thetherapeutic transgene product is expressed in vivo by the neural orpre-neural cells, thereby inhibiting development of the neurologicaldisease or disorder.

As noted above, transformation can be effected either in vivo or ex vivo(i.e., using differentiated neural cells, neural stem cells, orembryonic stem cells which differentiate into neural cells). A preferredin vivo route of delivery is administering the HSV amplicon particlesdirectly to the neural cells which are to be treated using, e.g., thedelivery routes listed above. The neuronal disease or disorder whosedevelopment can be inhibited, and the therapeutic transgene productassociated therewith, are those which are listed above by way ofexample.

In addition to the foregoing uses described, the HSV amplicon particlesof the present invention can also be used for delivery of othertherapeutic transgenes as reported previously in the literature (i.e.,using other vectors or HSV-derived vectors prepared according tohelper-virus procedures or previously reported helper virus-freeprocedures). By way of example, Kutubuddin et al., “Eradication ofPre-Established Lymphoma Using Herpes Simplex Virus Amplicon Vectors,”Blood 93(2):643-654 (1999), which is hereby incorporated by reference inits entirety, reports on the use of helper virus-prepared HSV ampliconparticles which transduce CD80 or RANTES, eliciting a protective immuneresponse to pre-established lymphoma and generating tumor-specificcytotoxic T-cells immunity and immunologic memory.

EXAMPLES

The following examples are provided to illustrate an embodiment of thepresent invention but is by no means intended to limit its scope.

Materials & Methods

Cell Culture

Baby hamster kidney (BHK) cells were maintained as described before (Luand Federoff, “Herpes simplex virus type 1 amplicon vectors withglucocorticoid-inducible gene expression,” Hum. Gene Ther. 6:421-430(1995), which is hereby incorporated by reference in its entirety). TheNIH-3T3 mouse fibroblast cell line was originally obtained from AmericanType Culture Collection and maintained in Dulbecco's modified Eaglemedium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin,and streptomycin.

Plasmid Construction

The HSVPrPUC/CMVegfp amplicon plasmid was constructed by cloning the0.8-kb cytomegalovirus (CMV) immediate early promoter and 0.7-kbenhanced green fluorescent protein cDNA (Clontech, Inc.) into the BamHIrestriction enzyme site of the pHSVPrPUC amplicon vector.

A 3.5 kb Hpa I/Hind III fragment encompassing the UL41 (vhs) openreading frame and its 5′ and 3′ transcriptional regulatory elements wasremoved from cos56 (Cunningham and Davison, “A cosmid-based system forconstruction mutants of herpes simplex type 1,” Virology, 197:116-124(1993), which is hereby incorporated by reference in its entirety) andcloned into pBSKSII (Stratagene, Inc.) to create pBSKS(vhs).

For construction of pGRE₅vp16, the VP16 coding sequence was amplified byPCR from pBAC-V2 using gene-specific oligonucleotides that possess EcoRIand HindIII restriction enzyme sequences that facilitates cloning intothe pGRE₅-2 vector (Mader and White, “A steroid-inducible promoter forthe controlled overexpression of cloned genes in eukaryotic cells,”Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993), which is herebyincorporated by reference in its entirety). The oligonucleotidepossessing the EcoRI site has a nucleotide sequence according to SEQ IDNo: 6 as follows:cggaattccg caggttttgt aatgtatgtg ctcgt  35The oligonucleotide possessing the HindIII site has a nucleotidesequence according to SEQ ID No: 7 as follows:ctccgaagct taagcccgat atcgtctttc ccgtatca  38

Helper Virus-Free Amplicon Packaging

On the day prior to transfection, 2×1⁶ BHK cells were seeded on a 60-mmculture dish and incubated overnight at 37° C. For cosmid-basedpackaging: The day of transfection, 250 μl Opti-MEM (Gibco-BRL,Bethesda, Md.), 0.4 μg of each of the five cosmid DNAs and 0.5 μgamplicon vector DNA with or without varying amounts of pBSKS(vhs)plasmid DNA were combined in a sterile polypropylene tube (Fraefel etal., “Helper virus-free transfer of herpes simplex virus type 1 plasmidvectors into neural cells,” J. Virol. 70:7190-7197 (1996), which ishereby incorporated by reference in its entirety). For BAC-basedpackaging: 250 μl Opti-MEM (Gibco-BRL, Bethesda, Md.), 3.5 μg of pBAC-V2DNA and 0.5 μg amplicon vector DNA with or without varying amounts ofpBSKS(vhs) plasmid DNA were combined in a sterile polypropylene tube(Stavropoulos and Strathdee, “An enhanced packaging system forhelper-dependent herpes simplex virus vectors,” J. Virol., 72:7137-43(1998), which is hereby incorporated by reference in its entirety). Theprotocol for both cosmid- and BAC-based packaging was identical from thefollowing step forward. Ten microliters of Lipofectamine Plus Reagent(Gibco-BRL) were added over a 30-second period to the DNA mix andallowed to incubate at RT for 20 min. In a separate tube, 15 μlLipofectamine (Gibco-BRL) were mixed with 250 μl Opti-MEM. Following the20-min incubation, the contents of the two tubes were combined over a1-min period, and incubated for an additional 20 min at RT. During thesecond incubation, the medium in the seeded 60-mm dish was removed andreplaced with 2 ml Opti-MEM. The transfection mix was added to the flaskand allowed to incubate at 37° C. for 5 hrs. The transfection mix wasthen diluted with an equal volume of DMEM plus 20% FBS, 2%penicillin/streptomycin, and 2 mM hexamethylene bis-acetamide (HMBA),and incubated overnight at 34° C. The following day, medium was removedand replaced with DMEM plus 10% FBS, 1% penicillin/streptomycin, and 2mM HMBA. The packaging flask was incubated an additional 3 days andvirus harvested and stored at −80° C. until purification. Viralpreparations were subsequently thawed, sonicated, and clarified bycentrifugation (3000×g, 20 min.). Viral samples were stored at −80° C.until use. For packaging experiments examining the effect of VP16 onamplicon titers, the cells plated for packaging were first allowed toadhere to the 60-mm culture dish for 5 hours and subsequentlytransfected with pGRE₅vp16 using the Lipofectamine reagent as describedabove. Following a 5-hr incubation, the transfection mix was removed,complete medium (DMEM plus 10% FBS, 1% penicillin/streptomycin) wasadded, and the cultures were incubated at 37° C. until the packagingco-transfection step the subsequent day.

Viral Titering

Amplicon titers were determined by counting the number of cellsexpressing enhanced green fluorescent protein (HSVPrPUC/CMVegfpamplicon) or β-galactosidase (HSVlac amplicon). Briefly, 10 μl ofconcentrated amplicon stock was incubated with confluent monolayers(2×10⁵ expressing particles) of NIH 3T3 cells plated on glasscoverslips. Following a 48-hr incubation, cells were either fixed with4% paraformaldehyde for 15 min at RT and mounted in Moiwol forfluorescence microscopy (eGFP visualization), or fixed with 1%glutaraldehyde and processed for X-gal histochemistry to detect the lacZtransgene product Fluorescent or X-gal-stained cells were enumerated,expression titer calculated, and represented as either green-formingunits per ml (gfu/ml) or blue-forming units per ml (bfu/ml),respectively.

TaqMan Quantitative PCR System

To isolate total DNA for quantitation of amplicon genomes in packagedstocks, virions were lysed in 100 mM potassium phosphate pH 7.8 and 0.2%Triton X-100. Two micrograms of genomic carrier DNA was added to eachsample. An equal volume of 2× Digestion Buffer (0.2 M NaCl, 20 mMTris-Cl pH 8, 50 mM EDTA, 0.5% SDS, 0.2 mg/ml proteinase K) was added tothe lysate and the sample was incubated at 56° C. for 4 hrs. Sampleswere processed further by one phenol:chloroform, one chloroformextraction, and a final ethanol precipitation. Total DNA was quantitatedand 50 ng of DNA was analyzed in a PE7700 quantitative PCR reactionusing a designed lacZ-specific primer/probe combination multiplexed withan 18S rRNA-specific primer/probe set. The lacZ probe sequence (SEQ IDNo: 8) was as follows:6FAM-accccgtacg tcttcccgag cg-TAMRA  22where 6FAM is a (6-carboxyfluorescein) conjugated dye and TAMRA is a(6-carboxytetramethylrhodamine) conjugated quencher. The lacZ senseprimer sequence (SEQ ID No: 9) was as follows:gggatctgcc attgtcagac at  22The lacZ antisense primer sequence (SEQ ID No: 10) was as follows:tggtgtgggc cataattcaa  20The 18S rRNA probe sequence (SEQ ID No: 11) was as follows:JOE-tgctggcacc agacttgccc tc-TAMRA  22where JOE is a (6-carboxy4′,5′-dichloro-2′,7′-dimethoxyfluorescein)conjugated dye. The 18S sense primer sequence (SEQ ID No: 12) was asfollows:cggctaccac atccaaggaa  20The 18S antisense primer sequence (SEQ ID No: 13) was as follows:gctggaatta ccgaggct  18

Each 25-μl PCR sample contained 2.5 μl (50 ng) of purified DNA, 900 nMof each primer, 50 nM of each probe, and 12.5 μl of 2× Perkin-ElmerMaster Mix. Following a 2-min 50° C. incubation and 2-min 95° C.denaturation step, the samples were subjected to 40 cycles of 95° C. for15 sec. and 60° C. for 1 min. Fluorescent intensity of each sample wasdetected automatically during the cycles by the Perkin-Elmer AppliedBiosystem Sequence Detector 7700 machine. Each PCR run included thefollowing: no-template control samples, positive control samplesconsisting of either amplicon DNA (for lacZ) or cellular genomic DNA(for 18S rRNA), and standard curve dilution series (for lacZ and 18S).Following the PCR run, “real-time” data were analyzed using Perkin-ElmerSequence. Detector Software version 1.6.3 and the standard curves.Precise quantities of starting template were determined for eachtitering sample and results were expressed as numbers of vector genomesper ml of original viral stock.

Western Blot Analysis

BHK cell monolayers (2×10⁶ cells) transfected with varying packagingcomponents were lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC,0.5% SDS, and 50 mM Tris-Cl, pH 8). Equal amounts of protein wereelectrophoretically separated on a 10% SDS-PAGE gel and transferred to aPVDF membrane. The resultant blot was incubated with an anti-VP16monoclonal antibody (Chemicon, Inc.), and specific VP16 immunoreactiveband visualized using an alkaline phosphatase-based chemiluminescentdetection kit (ECL).

Stereotactic Infections

Mice were anesthetized with Avertin at a dose of 0.6 ml per 25 g bodyweight. After positioning in an ASI murine stereotactic apparatus, theskull was exposed via a midline incision, and burr holes were drilledover the following coordinates (bregma, +0.5 mm; lateral −2.0 mm; anddeep, −3.0 mm) to target infections to the striatum. A 33 GA steelneedle was gradually advanced to the desired depth, and 3 μl ofHSVPrPUC/CMVegfp virus was infused via a nicroprocessor-controlled pumpover 10 minutes (UltraMicroPump, World Precision Instruments, SarasotaSprings, Fla.). The injector unit was mounted on a precision smallanimal stereotaxic frame (ASI Instruments, Warren, Mich.)micromanipulator at a 90° angle using a mount for the injector. Viralinjections were performed at a constant rate of 300 nl/min. The needlewas removed slowly over an additional 10-minute period.

Tissue Preparation and GFP Visualization

Infected mice were anesthetized four days later, a catheter was placedinto the left ventricle, and intracardiac perfusion was initiated with10 ml of heparinized saline (5,000 U/L saline) followed by 60 ml ofchilled 4% PFA. Brains were extracted and postfixed for 1-2 hours in 4%PFA at 4° C. Subsequently, brains were cryoprotected in a series ofsucrose solutions with a final solution consisting of a 30% sucroseconcentration (w/v) in PBS. Forty micron serial sections were cut on asliding microtome (Micron/Zeiss, Thomwood, N.Y.) and stored in acryoprotective solution (30% sucrose (w/v), 30% ethylene glycol in 0.1 Mphosphate buffer (pH 7.2)) at −20° C. until processed for GFPvisualization. Sections were placed into Costar net wells (VWR,Springfield, N.J.) and incubated for 2 hrs in 0.1 M Tris buffered saline(TBS) (pH=7.6). Upon removal of cryoprotectant, two additional 10 minwashes in 0.1 M TBS with 0.25% Triton X-100 (Sigma, St. Louis, Mo.) wereperformed. Sections were mounted with a fine paint brush onto subbedslides, allowed to air dry, and mounted with an aqueous mounting media,Mowiol. GFP-positive cells were visualized with a fluorescent microscope(Axioskop, Zeiss, Thornwood, N.Y.) utilizing a FITC cube (ChromaFilters, Brattleboro, Vt.). All images used for morphological analyseswere digitally acquired with a 3-chip color CCD camera at 200×magnification (DXC-9000, Sony, Montvale, N.J.).

Morphological Analyses

Cell counts were performed on digital images acquired within 24 hrs ofmounting. At the time of tissue processing coronal slices were storedserially in three separate compartments. All compartments were processedfor cell counting and GFP(+) cell numbers reflect cell counts throughoutthe entire injection site. All spatial measurements were acquired usingan image analysis program (Image-Pro Plus, Silver Spring, Md.) at afinal magnification of 200×. Every section was analyzed using identicalparameters in three different planes of focus throughout the section toprevent repeated scoring of GFP(+) cells. Each field was analyzed by acomputer macro to count cells based on the following criteria: objectarea, image intensity (fluorescent signal) and plane of focus. Onlycells in which the cell body was unequivocally GFP(+) and nucleusclearly defined were counted. Every section that contained aGFP-positive cell was counted. In addition, a watershed separationtechnique was applied to every plane of focus in each field to delineateoverlapping cell bodies. The watershed method is an algorithm that isdesigned to erode objects until they disappear, then dilates them againsuch that they do not touch.

Example 1 Effect of Amplicon Co-Transfection With Vhs Vector

To determine if introduction of vhs into the packaging scheme couldincrease amplicon titers and quality, a genomic segment of the UL41 genewas cloned into pBluescript and the resulting plasmid (pBSKS(vhs)) wasintroduced into co-transfection protocols to provide vhs in trans. Thegenomic copy of UL41 contained the transcriptional regulatory region andflanking cis elements believed to confer native UL41 gene expressionduring packaging. When pBSKS(vhs) was added to the packaging protocolsfor production of a β-galactosidase (lacZ)-expressing amplicon (HSVlac),a maximum of 10-fold enhanced amplicon expression titers was observedfor both cosmid- and BAC-based strategies (FIGS. 6A and B,respectively). As observed previously, the expression titers for HSVlacvirus produced by the BAC-based method were approximately 500- to1000-fold higher than stocks produced using the modified cosmid set.Even though a large disparity existed between the differently preparedstocks, the effect of additionally expressed vhs on amplicon titers wasanalogous.

The punctate appearance of reporter gene product (pseudotransduction), aphenomenon associated with first-generation helper virus-free stocks,was drastically diminished in vitro when vhs was included in BAC-basedpackaging of an enhanced green fluorescent (GFP)-expressing virus(HSVPrPUC/CMVegfp) (FIGS. 7C-D). Pseudotransduction was not observed, aswell, for cosmid-packaged amplicon stocks prepared in the presence ofvhs.

To assess the ability of the improved amplicon stocks to mediate genedelivery in vivo, 3 μl of BAC-packaged HSVPrPUC/CMVegfp virus preparedin the absence or presence of pBSKS(vhs) was injected stereotacticallyinto the striata of C57BL/6 mice. Four days following infection, animalswere sacrificed and analyzed for GFP-positive cells present in thestriatum (FIGS. 7E-F). The numbers of cells transduced byHSVPrPUC/CMVegfp prepared in the presence of vhs were significantlyhigher than in animals injected with stocks produced in the absence ofvhs (FIG. 7G). In fact, it was difficult to definitively identifyGFP-positive cells in animals transduced with vhs(−) amplicon stocks.

The mechanism by which vhs expression resulted in higher apparentamplicon titers in helper virus-free packaging could be attributed toone or several properties of vhs. The UL41 gene product is a componentof the viral tegument and could be implicated in structural integrity,and its absence could account for the appearance of punctate geneproduct material following transduction. For example, the viralparticles may be unstable as a consequence of lacking vhs. Thus,physical conditions, such as repeated freeze-thaw cycles or long-termstorage, may have led to inactivation or destruction of vhs-lackingvirions at a faster rate than those containing vhs.

The stability of HSVPrPUC/CMVegfp packaged via the BAC method in thepresence or absence of vhs was analyzed initially with a series ofincubations at typically used experimental temperatures. Viral aliquotsfrom prepared stocks of HSVPrPUC/CMVegfp were incubated at 4, 22, or 37°C. for periods up to three hours. Virus recovered at time points 0, 30,60, 120, and 180 minutes were analyzed for their respective expressiontiter on NIH 3T3 cells. The rates of decline in viable ampliconparticles, as judged by their ability to infect and express GFP, did notdiffer significantly between the vhs(+) and vhs(−) stocks (FIGS. 8A-C).Another condition that packaged amplicons encounter during experimentalmanipulation is freeze-thaw cycling. Repetitive freezing and thawing ofvirus stocks is known to diminish numbers of viable particles, andpotentially the absence of vhs in the tegument of pBAC-V2 packagedamplicons leads to sensitivity to freeze fracture. To test thispossibility, viral aliquots were exposed to a series of four freeze-thawcycles. Following each cycle, samples were removed and titered for GFPexpression on NIH 3T3 cells as described previously. At the conclusionof the fourth freeze-thaw cycle, the vhs(−) HSVPrPUC/CMVegfp stockexhibited a 10-fold diminution in expression titers as opposed to only a2-fold decrease for vhs(+) stocks (FIG. 8D). This observation suggeststhat not only do vhs(+) stocks have increased expression titers, but thevirions are more stable when exposed to temperature extremes, asdetermined by repetitive freeze-thaw cycling.

Wild-type HSV virions contain multiple regulatory proteins that preparean infected host cell for virus propagation. One of these virallyencoded regulators, which is localized to the tegument, is vhs. The UL41gene-encoded vhs protein exhibits an essential endoribonucleolyticcleavage activity during lytic growth that destabilizes both cellularand viral mRNA species (Smibert et al., “Identification andcharacterization of the virion-induced host shutoff product of herpessimplex virus gene UL41,” J. Gen. Virol., 73:467-470 (1992), which ishereby incorporated by reference in its entirety). Vhs-mediatedribonucleolytic activity appears to prefer the 5′ ends of mRNAs over 3′termini, and the activity is specific for mRNA, as vhs does not act uponribosomal RNAs (Karr and Read, “The virion host shutoff function ofherpes simplex virus degrades the 5′ end of a target mRNA before the 3′end,” Virology, 264:195-204 (1999), which is hereby incorporated byreference in its entirety). Vhs also serves a structural role in virusparticle maturation as a component of the tegument HSV isolates thatpossess disruptions in UL41 demonstrate abnormal regulation of IE genetranscription and significantly lower titers than wild-type HSV-1 (Readand Frenkel, “Herpes simplex virus mutants defective in thevirion-associated shutoff of host polypeptide synthesis and exhibitingabnormal synthesis of α (immediate early) viral polypeptides,” J. Virol.46:498-512 (1983), which is hereby incorporated by reference in itsentirety), presumably due to the absence of vhs activity. Therefore,because vhs is essential for efficient production of viable wild-typeHSV particles, it likely plays a similarly important role in packagingof HSV-1-derived amplicon vectors.

The term “pseudotransduction” refers to virion expression-independenttransfer of biologically active vector-encoded gene product to targetcells (Liu et al., “PseudotRansduction of hepatocytes by usingconcentrated pseudotyped vesicular stomatitis virus G glycoprotein(VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors:comparison of VSV-G and amphotrophic vectors for hepatic gene transfer,”J. Virol., 70: 2497-2502 (1996); Alexander et al., “Transfer ofcontaminants in adeno-associated virus vector stocks can mimictransduction and lead to artifactual results,” Hum. Gene Ther.8:1911-1920 (1997); Yu et al., “High efficiency in vitro gene transferinto vascular tissues using a pseudotyped retroviral vector withoutpseudotransduction,” Gene Ther., 6:1876-1883 (1999), which are herebyincorporated by reference in their entirety). This phenomenon wasoriginally described with retrovirus and adeno-associated virus vectorstocks and was shown to result in an overestimation of gene transferefficiencies. β-galactosidase and alkaline phosphatase are two commonlyexpressed reporter proteins that have been implicated inpseudotransduction, presumably due to their relatively high enzymaticstability and sensitivity of their respective detection assays(Alexander et al., “Transfer of contaminants in adeno-associated virusvector stocks can mimic transduction and lead to artifactual results,”Hum. Gene Ther. 8:1911-1920 (1997), which is hereby incorporated byreference in its entirety). Stocks of β-galactosidase-expressing HSVlacand GFP-expressing HSVPrPUC/CMVegfp exhibited high levels ofpseudotransduction when packaged in the absence of vhs. Upon addition ofvhs to the previously described helper virus-free packaging protocols(Fraefel et al., “Helper virus-free transfer of herpes simplex virustype 1 plasmid vectors into neural cells,” J. Virol. 70:7190-7197(1996); Stavropoulos and Strathdee, “An enhanced packaging system forhelper-dependent herpes simplex virus vectors,” J. Virol., 72:7137-43(1998), which are hereby incorporated by reference in their entirety), a10-fold increase in expression titers and concomitant decrease inpseudotransduction were observed in vitro.

Vhs-mediated enhancement of HSV amplicon packaging was even more evidentwhen stocks were examined in vivo. GFP-expressing cells in animalstransduced with vhs(+) stocks were several hundred-fold greater innumber than in animals receiving vhs(−) stocks. This could have been dueto differences in virion stability, where decreased particle stabilitycould have led to release of co-packaged reporter gene product observedin the case of vhs(−) stocks. Additionally, the absence of vhs may haveresulted in packaging of reporter gene product into particles thatconsist of only tegument and envelope (Rixon et al., “Assembly ofenveloped tegument structures (L particles) can occur independently ofvirion maturation in herpes simplex virus type 1-infected cells,” J.Gen. Virol., 73:277-284 (1992), which is hereby incorporated byreference in its entirety). Release of co-packaged reporter gene productin either case could potentially activate a vigorous immune response inthe CNS, resulting in much lower than expected numbers ofvector-expressing cells.

Interestingly, the HSV-encoding cosmid set harbored an intact UL41 genelocus (Cunningham and Davison, “A cosmid-based system for constructionmutants of herpes simplex type 1,” Virology 197:116-124 (1993), which ishereby incorporated by reference), while the BAC reagent that wasutilized for helper virus-free packaging did not because of a disruptionintroduced during its initial construction (Stavropoulos and Strathdee,“An enhanced packaging system for helper-dependent herpes simplex virusvectors,” J. Virol., 72:713743 (1998), which is hereby incorporated byreference). Expression of vhs via a co-transfected plasmid containingthe entire UL41 gene plus its cognate transcriptional regulatory regionsresulted in pronounced increases in packaged amplicon produced viaeither cosmid- or BAC-based method. For BAC-based packaging, theexplanation appears rather clear: vhs is not expressed due to disruptionof the UL41 locus, and therefore, inclusion of a vhs expression plasmidresults in a more productive packaging. In the case for cosmid-basedpackaging, the copy number of the co-transfected vhs-encoding plasmidgreatly exceeded the number of vhs transcription units present in thecosmid set. This likely led to a more rapid accumulation of vhs duringthe early stages of packaging. Additionally, because the cosmid set isbelieved to undergo recombination of its overlapping homologous regionsto produce a HSV genome-sized unit following introduction into thepackaging cell, perhaps viral gene expression is delayed (Cunningham andDavison, “A cosmid-based system for construction mutants of herpessimplex type 1,” Virology, 197:116-124 (1993), which is herebyincorporated by reference). As a result, amplicon propagation cannotoptimally initiate.

The resulting HSV amplicon particles were also examined by scanningelectron micrography using a standard negative staining technique(Monroe and Brandt, “Rapid semiquantitative method for screening largenumbers of virus samples by negative staining electron microscopy,” ApplMicrobiol 20(2):259-62 (1970), which is hereby incorporated by referencein its entirety). As shown in FIG. 13, the HSV amplicon particles,denoted by arrows, are substantially smaller in size than the 173 nmreference spheres and rather heterogeneous in structure. In contrast,helper virus-containing stocks are characterized by the production ofHSV amplicon particles which are approximately 150 nm in size and morehomogeneous in shape. Thus, the HSV amplicon particles of the presentinvention are physically different from previously known helpervirus-prepared HSV amplicon particles.

Example 2 Effect of VP16 Expression in Host Cells Prior to AmpliconCo-Transfection

The native HSV genome enters the host cell with several viral proteinsbesides vhs, including the strong transcriptional activator VP16. Oncewithin the cell, VP16 interacts with cellular transcription factors andHSV genome to initiate immediate-early gene transcription. Under helpervirus-free conditions, transcriptional initiation of immediate-earlygene expression from the HSV genome may not occur optimally, thusleading to lower than expected titers. To address this issue, a VP16expression construct was introduced into packaging cells prior tocosmid/BAC, amplicon, and pBSKS(vhs) DNAs, and resultant amplicon titerswere measured. To achieve regulated expression aglucocorticoid-controlled VP16 expression vector was used (pGRE₅vp16).

The pGRE₅vp16 vector was introduced into the packaging cells 24 hoursprior to transfection of the regular packaging DNAs. HSVlac was packagedin the presence or absence of vhs and/or VP16 and resultant ampliconstocks were assessed for expression titer. Some packaging culturesreceived 100 nM dexamethasone at the time of pGRE₅vp16 transfection tostrongly induce VP16 expression; others received no dexamethasone.Introduction of pGRE₅vp16 in an uninduced (basal levels) or inducedstate (100 nM dexamethasone) had no effect on HSVlac titers when vhs wasabsent from the cosmid- or BAC-based protocol (FIGS. 9A-B). In thepresence of vhs, addition of pGRE₅vp16 led to either a two- or five-foldenhancement of expression titers over those of stocks packaged with onlyvhs (cosmid- and BAC-derived stocks, respectively; FIGS. 9A-B). Theeffect of “uninduced” pGRE₅vp16 on expression titers suggested that VP16expression was occurring in the absence of dexamethasone. To demonstratethis, Western blot analysis with a VP16-specific monoclonal antibody wasperformed using lysates prepared from BHK cells transfected with thevarious packaging components. Cultures transfected withpGRE₅vp16/BAC/pBSKS(vhs) in the absence of dexamethasone did show VP16levels intermediate to cultures transfected either with BAC alone(lowest) or those transfected with pGRE₅vp16/BAC/pBSKS(vhs) in thepresence of 100 nM dexamethasone (highest)(FIG. 9C).

VP16-mediated enhancement of packaged amplicon expression titers couldbe due to increased DNA replication and packaging of amplicon genomes.Conversely, the additional VP16 that is expressed via pGRE₅vp16 could beincorporated into virions and act by increasing vector-directedexpression in transduced cells. To test the possibility that VP16 isacting by increasing replication in the packaging cells, concentrationsof vector genomes in BAC-derived vector stocks were determined. HSVlacstocks produced in the presence or absence of vhs and/or VP16 wereanalyzed using a “real-time” quantitative PCR method. The concentrationof vector genome was increased two-fold in stocks prepared in thepresence of VP16 and this increase was unaffected by the presence of vhs(FIG. 10). VP16 expression was induced with 100 nM dexamethasonetreatment at varying time points prior to introduction of the packagingcomponents. Dexamethasone-induced production of VP16 prior totransfection of the packaging components did not appear to enhanceamplicon titers over that observed with basal pGRE₅vp16-mediatedexpression (FIG. 11). This suggests that low levels of VP16 aresufficient to enhance amplicon packaging in the presence of vhs.

Pre-loading of packaging cells with low levels of the potent HSVtranscriptional activator VP16 led to a 2- to 5-fold additional increasein amplicon expression titers only in the presence of vhs for cosmid-and BAC-based packaging systems, respectively. This observationindicates the transactivation and structural functions of VP16 were notsufficient to increase viable viral particle production when vhs wasabsent, and most likely led to generation of incomplete virionscontaining amplicon genomes as detected by quantitative PCR. When vhswas present for viral assembly, however, VP16-mediated enhancement ofgenome replication led to higher numbers of viable particles formed. Theeffect of VP16 on expression titers was not specific to ampliconspossessing the immediate-early 4/5 promoter of HSV, as amplicons withother promoters were packaged to similar titers in the presence of VP16and vhs.

VP16 is a strong transactivator protein and structural component of theHSV virion (Post et al., “Regulation of alpha genes of herpes simplexvirus: expression of chimeric genes produced by fusion of thymidinekinase with alpha gene promoters,” Cell, 24:555-565 (1981), which ishereby incorporated by reference). VP16-mediated transcriptionalactivation occurs via interaction of VP16 and two cellular factors,Oct-1 (O'Hare and Goding, “Herpes simplex virus regulatory elements andthe immunoglobulin octamer domain bind a common factor and are bothtargets for virion transactivation,” Cell, 52:435-445 (1988); Preston etal., “A complex formed between cell components and an HSV structuralpolypeptide binds to a viral immediate early gene regulatory DNAsequence,” Cell, 52:425-434 (1988); Stern et al., “The Oct-1homoeodomain directs formation of a multiprotein-DNA complex with theHSV transactivator VP16,” Nature. 341:624-630 (1989), which are herebyincorporated by reference in their entirety) and HCF (Wilson et al.,“The VP16 accessory protein HCF is a family of polypeptides processedfrom a large precursor protein,” Cell 74:115-125 (1993); Xiao andCapone, “A cellular factor binds to the herpes simplex virus type 1transactivator Vmw65 and is required for Vmw65-dependent protein-DNAcomplex assembly with Oct-1,” Mol. Cell Biol., 10:4974-4977 (1990),which are hereby incorporated by reference in their entirety), andsubsequent binding of the complex to TAATGARAT elements found within HSVIE promoter regions (O'Hare, “The virion transactivator of herpessimplex virus,” Semin. Virol. 4:145-155 (1993), which is herebyincorporated by reference). This interaction results in robustup-regulation of IE gene expression. Neuronal splice-variants of therelated Oct-2 transcription factor have been shown to block IE geneactivation via binding to TAATGARAT elements (Lillycrop et al., “Theoctamer-binding protein Oct-2 represses HSV immediate-early genes incell lines derived from latently injectable sensory neurons,” Neuron,7:381-390 (1991), which is hereby incorporated by reference), suggestingthat cellular transcription factors may also play a role in limiting HSVlytic growth.

The levels of VP16 appear to be important in determining its effect onexpression titers. Low, basal levels of VP16 (via uninduced pGRE₅vp16)present in the packaging cell prior to introduction of the packagingcomponents induced the largest effect on amplicon expression titers.Conversely, higher expression of VP16 (via dexamethasone-inducedpGRE₅vp16) did not enhance virus production to the same degree and mayhave, in fact, abrogated the process. The presence of glucocorticoids inthe serum components of growth medium is the most likely reason for thislow-level VP16 expression, as charcoal-stripped sera significantlyreduces basal expression from this construct. Perhaps only a low levelor short burst of VP16 is required to initiate IE gene transcription,but excessive VP16 leads to disruption of the temporal progressionthrough the HSV lytic cycle, possibly via inhibition of vhs activity.Moreover, evidence has arisen to suggest vhs activity is downregulatedby newly synthesized VP16 during the HSV lytic cycle, thereby allowingfor accumulation of viral mRNAs after host transcripts have beendegraded (Smibert et al., “Herpes simplex virus VP16 forms a complexwith the virion host shutoff protein vhs,” J. Virol. 68(4):233-236(1994); Lam et al., “Herpes simplex virus VP16 rescues viral mRNA fromdestruction by the virion host shutoff function,” EMBO J., 15:2575-2581(1996), which are hereby incorporated by reference in their entirety).Therefore, a delicate regulatory protein balance may be required toattain optimal infectious particle propagation. Additionally, the 100-nMdexamethasone treatment used to induce VP16 expression may have adeleterious effect on cellular gene activity and/or interfere withreplication of the OriS-containing amplicon genome in packaging cells.High levels of dexamethasone have been shown previously to repress HSV-1OriS-dependent replication by an unknown mechanism (Hardwicke andSchaffer, “Differential effects of nerve growth factor and dexamethasoneon herpes simplex virus type 1 oriL- and oriS-dependent DNA replicationin PC12 cells,” J. Virol., 71:3580-3587 (1997), which is herebyincorporated by reference in its entirety).

Example 3 Examination of Amplicon Cytotoxicity

There is a possibility that addition of viral proteins, like vhs andVP16, to the packaging process may lead to vector stocks that areinherently more cytotoxic. The amplicon stocks described above wereexamined for cytotoxicity using a lactate dehydrogenase (LDH)release-based cell viability assay. Packaged amplicon stocks were usedto transduce NIH 3T3 cells and 48 hours following infection, viabilityof the cell monolayers was assessed by the LDH-release assay. Ampliconstocks produced in the presence of vhs and VP16 displayed lesscytotoxicity on a per virion basis than stocks packaged using thepreviously published BAC-based protocol (FIG. 12) (Stavropoulos andStrathdee, “An enhanced packaging system for helper-dependent herpessimplex virus vectors,” J. Virol., 72:7137-43 (1998), which is herebyincorporated by reference in its entirety)).

Ectopic expression of vhs and VP16 did not lead to amplicon stocks thatexhibited higher cytotoxicity than helper virus-free stocks prepared inthe traditional manner when examined by an LDH-release assay. Stocksprepared by the various methods were equilibrated to identicalexpression titers prior to exposure to cells. The heightenedcytotoxicity in stocks produced in the absence of vhs and/or VP16 mayreflect that larger volumes of these stocks were required to obtainsimilar expression titers as the vhs/VP16-containing samples or thelevels of defective particles in the former may be significantly higher.Contaminating cellular proteins that co-purify with the ampliconparticles are most likely higher in concentration in the traditionalstocks, and probably impart the higher toxicity profiles observed.

Example 4 Comparative Analysis of Helper Virus-Free HSV AmpliconParticles and Helper Virus HSV Amplicon Particles

Helper virus-free HSV amplicon particles were prepared as describedabove in Example 1 and helper virus-containing HSV amplicon particleswere prepared according to known procedures.

Two-dimensional gel analyses were performed on stocks containing thehelper virus-free (HVF) virion particles (FIG. 14) and helpervirus-containing (HVC) virion particles (FIG. 15) to determinedifferences in their protein composition. Virion particles from bothhelper virus-containing and helper virus-free amplicon stocks werepurified by ultracentrifugation on a 30%/60% discontinuous sucrosegradient. Bands containing viral particles were extracted from thegradient at the 30%/60% interface and stored at −80° C. until 2-D gelanalyses were performed. Prior to gel analyses, protein concentrationwas determined by the Bradford assay and 100 μg of each sample wasresuspended in urea sample buffer (9.5 M ultrapure urea, 2% w/v NonidetP-40, 5% beta-mercaptoethanol, and 2% ampholines consisting of 1.6% pH5-7 and 0.4% pH 3.5-10). Fifty μg of each sample was run 2X's on 2-Dgels (ampholines pH of 3.5-10), the gels were silver-stained, digitized,and analyzed by comparison of 2-D patterns and spot intensity of helpervirus-containing vs. helper virus-free amplicon stocks.

As shown in Table 2 below, the reference spot number, pI, and molecularweight (daltons) are given for polypeptide spots analyzed in the samplesobtained from the stocks of HVF and HVC virion particles. Also indicatedin Table 2 are the fold increase or decrease (difference) of thepolypeptides for gel bands from the two samples. Spot percentages werecalculated as individual spot density divided by total density of allmeasured spots. The difference is calculated from spot density asfollows:

${Difference} = {\frac{\left( {1 - {{Spot}\mspace{14mu}{Percentage}\mspace{14mu}{of}\mspace{14mu}{HVC}}} \right)}{\left( {{Spot}\mspace{14mu}{Percentage}\mspace{14mu}{of}\mspace{14mu}{HVF}} \right)} \times {- 100}}$A significant increase in the polypeptide spot density is considered tobe a difference ≧+300, where a significant decrease in the polypeptidespot density is considered to be a difference ≦−67. Significantlyincreased and decreased polypeptide spots are highlighted (outlined) inFIGS. 16A-B and 17A-C, respectively.

TABLE 2 Summary of Two-Dimensional Gel Protein Analysis HelperVirus-Free Helper Virus-Containing Spot No. pI MW Spot Percent SpotPercent Difference 1 6.04 150,730 0.24 0.05 −79 2 6.14 121,290 0.02 0.09341 3 5.94 103,956 0.61 0.01 −99 4 5.74 96,220 0.34 0.17 −49 5 6.0293,124 0.07 0.03 −55 6 5.1 92,212 0.71 0.36 −49 7 5.59 89,821 0.00 0.1866661 8 5.6 87,909 0.02 0.06 220 9 6.28 87,423 0.44 0.03 −93 10 5.4885,649 0.00 0.05 3970 11 5.92 83,910 0.96 0.14 −85 12 6.97 83,902 0.010.15 1032 13 6.59 83,729 0.18 0.01 −97 14 6.7 83,729 0.02 0.61 3080 155.53 79,043 5.94 0.99 −83 16 6.06 77,562 1.91 0.48 −75 17 5.68 77,3040.06 0.00 −100 18 5.76 76,957 0.19 0.00 −99 19 6.31 76,697 0.02 0.02 −820 5.98 90,963 0.63 3.27 421 21 6.4 74,967 0.78 7.29 840 22 7.19 74,7420.10 0.05 −53 23 5.89 72,089 0.09 0.01 −88 24 5.87 70,698 0.02 0.00 −9425 5.7 70,177 0.19 0.01 −94 26 7.08 70,482 0.03 0.09 235 27 5.36 68,0900.04 0.06 57 28 6.21 68,220 0.09 0.00 −99 29 6.29 67,874 0.05 0.03 −3830 6.67 67,406 0.01 0.25 2639 31 5.75 66,526 0.03 0.01 −76 32 7.3168,097 0.12 0.40 239 33 5.52 65,483 0.12 3.41 2693 34 6.08 65,279 2.040.19 −91 35 4.99 64,885 0.45 0.41 −9 36 7.39 66,052 0.02 0.11 375 377.48 64,007 0.00 0.32 14050 38 6.17 62,165 0.01 0.22 3946 39 6.22 61,4730.02 0.12 676 40 5.43 61,136 5.90 1.38 −77 41 5.96 61,136 3.24 2.28 −3042 6.3 61,127 0.27 0.46 69 43 6.42 61,784 0.16 0.11 −31 44 6.74 62,2860.06 0.06 −8 45 8.44 61,726 0.02 0.79 4651 46 5.61 59,227 0.02 0.02 −1247 6.48 58,874 0.52 0.22 −57 48 6.59 58,365 3.00 2.01 −33 49 5.28 57,5860.00 0.04 ++++ 50 5.71 57,586 0.13 0.02 −89 51 5.57 56,355 0.08 0.02 −7352 7.48 57,859 0.07 0.02 −68 53 5.02 55,634 0.04 0.20 366 54 8.08 57,4870.00 0.52 ++++ 55 6.76 55,915 0.00 0.06 33872 56 7.63 57,152 0.08 0.1581 57 6.83 55,786 0.00 0.12 9161 58 6.9 55,658 0.05 1.59 3038 59 5.4854,714 0.17 0.11 −38 60 7.1 56,317 0.01 0.10 1799 61 7.48 56,189 0.010.03 412 62 8.28 56,540 0.02 0.30 1849 63 5.01 53,293 0.01 0.14 2347 646.29 53,761 0.07 0.04 −42 65 7.09 54,647 0.06 0.05 −28 66 8.54 54,3661.44 0.39 −73 67 6.12 53,106 0.22 0.01 −98 68 6.68 53,208 0.10 0.11 1169 6.26 52,582 0.11 0.01 −92 70 5.57 51,842 2.29 0.48 −79 71 6.06 51,9260.07 0.00 −100 72 5.71 51,295 0.60 0.12 −80 73 6.58 51,403 0.25 0.11 −5874 6.12 50,615 0.02 0.04 160 75 5.05 49,049 0.31 0.02 −94 76 5.64 49,7900.07 0.07 8 77 7.06 51,693 0.05 0.00 −92 78 4.97 48,610 0.13 0.06 −57 795.59 49,380 0.06 0.09 44 80 8.68 50,067 0.05 0.01 −82 81 5.35 47,8760.09 0.01 −88 82 5.6 47,055 0.21 0.05 −75 83 5.16 45,244 0.23 0.06 −7484 8.79 47,487 0.15 0.40 167 85 8.66 47,344 0.06 0.08 34 86 5.67 45,9610.23 0.05 −81 87 6.67 47,149 0.00 0.85 33868 88 6.59 47,020 0.01 0.416309 89 6.26 46,289 0.21 0.02 −90 90 5.79 45,277 0.54 0.05 −91 91 6.4746,027 0.09 0.14 51 92 5.3 44,867 0.18 0.04 −77 93 8.15 46,934 0.13 0.10−26 94 7.39 46,426 0.00 0.07 10326 95 5.99 44,836 0.01 0.10 2005 96 7.1145,912 0.22 0.46 109 97 5.31 42,479 0.29 0.06 −80 98 7.48 44,885 0.010.11 1789 99 8.59 46,413 0.65 3.08 377 100 8.74 46,413 0.81 0.28 −65 1015.69 42,870 0.15 0.49 227 102 8.46 44,092 0.21 1.50 617 103 5.91 42,2961.30 2.59 99 104 6.14 42,491 0.05 0.07 63 105 5.33 41,888 1.11 0.81 −27106 7.39 45,972 0.02 0.08 409 107 6.29 42,187 0.11 0.02 −81 108 7.9742,453 1.24 0.92 −26 109 6.19 41,629 0.05 0.00 −100 110 7.74 42,193 0.160.49 211 111 7.46 41,779 0.16 0.01 −94 112 6.28 41,122 0.03 0.31 1004113 7.57 41,828 0.13 0.23 80 114 6.13 40,666 0.21 0.02 −92 115 8.7840,105 0.11 0.51 364 116 7.57 40,735 0.03 0.00 −96 117 5.39 39,543 0.100.01 −96 118 6.56 40,020 0.04 0.02 −61 119 5.33 39,135 0.05 0.00 −100120 7.49 40,094 0.17 0.13 −24 121 6.81 39,557 0.36 0.14 −60 122 7.6439,903 0.05 0.28 439 123 6.42 38,992 0.15 0.00 −100 124 6.38 38,536 0.130.10 −23 125 7.42 38,728 0.03 0.16 528 126 7.17 38,056 0.09 0.14 61 1275.6 36,841 0.01 0.07 1279 128 5.13 35,384 0.00 0.11 ++++ 129 5.98 36,1780.00 0.43 45454 130 7.52 37,007 0.21 0.00 −100 131 5.42 35,924 0.17 0.03−85 132 7.71 36,520 0.02 0.33 2141 133 5.62 35,516 0.03 0.15 473 1347.18 36,349 0.09 0.23 153 135 4.99 34,526 0.33 0.05 −84 136 5.98 35,3120.19 0.09 −50 137 6.39 35,645 0.03 0.66 1837 138 6.05 35,544 0.67 0.21−69 139 5.73 35,006 0.03 0.01 −76 140 5.02 33,830 0.53 0.21 −60 141 8.0435,162 3.36 7.90 135 142 7.55 35,584 0.05 0.35 553 143 6.57 34,883 0.040.47 1204 144 6 34,316 0.12 0.01 −92 145 6.06 34,479 0.03 0.14 396 1465.51 33,986 1.43 6.43 349 147 5.14 32,919 0.55 1.79 225 148 6.23 34,2250.32 0.18 −45 149 6.65 34,318 0.00 0.26 14364 150 6.54 33,855 0.06 0.0840 151 8.64 31,837 0.36 0.07 −79 152 6.07 32,856 0.24 0.48 96 153 6.2732,856 0.01 0.18 1132 154 8.83 31,493 0.39 0.13 −68 155 5.14 31,043 0.000.14 ++++ 156 5.29 31,794 0.01 0.16 2152 157 7.37 32,005 0.03 0.01 −72158 6.69 31,595 0.11 0.00 −100 159 6.08 31,233 0.04 0.33 697 160 6.5631,287 0.02 0.12 409 161 4.99 30,334 0.45 0.00 −99 162 5.72 30,214 0.010.17 3364 163 5.18 30,157 0.00 0.20 6047 164 6.52 30,619 0.00 0.03 23471165 5.63 30,329 0.05 0.38 686 166 6.46 29,610 0.43 0.15 −66 167 6.7529,643 0.33 0.17 −49 168 6.28 29,186 0.22 0.85 285 169 8.48 30,519 0.980.00 −100 170 6.07 28,978 1.99 0.43 −78 171 5.33 29,767 0.08 0.15 87 1727.88 28,993 0.33 0.09 −73 173 6.6 28,890 0.00 0.10 6034 174 7.45 28,8960.24 0.42 72 175 6.86 28,657 0.00 0.18 197412 176 7.23 28,654 0.00 0.39145023 177 6.98 28,210 0.05 0.01 −74 178 6.47 27,932 0.03 0.15 452 1796.64 27,992 0.26 0.88 247 180 6.24 27,822 0.05 0.02 −72 181 6.11 27,6390.55 0.00 −100 182 6.39 27,639 0.01 0.15 2823 183 6.74 27,677 0.05 0.0616 184 7.17 27,827 0.22 0.87 295 185 6.45 27,347 0.02 0.55 2959 186 7.6527,379 0.12 0.04 −68 187 6.29 26,871 0.22 0.09 −59 188 6.17 26,834 0.840.13 −84 189 5.36 26,421 0.37 0.02 −95 190 6.61 26,767 0.34 0.21 −38 1915 25,206 2.23 0.18 −92 192 5.69 26,122 0.08 0.90 978 193 5.95 26,0470.55 2.48 350 194 6.67 26,347 0.34 0.00 −100 195 6.57 26,312 0.13 0.00−99 196 5.33 25,186 0.00 0.09 2843 197 5.13 24,166 0.06 0.00 −97 1986.56 25,542 0.20 0.00 −99 199 5.91 24,812 0.35 0.04 −88 200 6.2 24,9310.32 0.03 −90 201 6.72 25,122 0.32 0.36 13 202 5.45 24,363 0.08 0.03 −63203 5.29 24,326 0.14 0.21 53 204 8.69 23,726 0.16 0.04 −78 205 9.3122,854 0.05 0.04 −28 206 7.81 24,487 0.30 0.49 67 207 6.58 24,212 0.330.12 −65 208 6.07 23,906 0.22 0.00 −100 209 9.06 22,562 0.12 0.04 −64210 7.55 24,313 0.08 0.04 −49 211 6.36 23,723 5.41 0.97 −82 212 8.4523,160 0.10 0.03 −71 213 7.68 23,407 0.07 0.02 −76 214 6.71 23,127 0.010.72 5995 215 6.09 22,699 0.29 0.00 −100 216 5.01 20,971 0.27 0.25 −5217 6.66 22,567 0.10 0.08 −20 218 5.42 21,406 2.46 1.17 −52 219 6.4321,381 0.20 0.17 −16 220 4.61 19,596 2.30 1.27 −45 221 6.62 21,063 0.190.08 −56 222 7.7 21,143 0.67 0.20 −71 223 8.81 19,769 0.23 0.05 −81 2246.18 20,173 1.76 0.06 −97 225 7.19 20,828 0.72 0.06 −92 226 6.78 20,5030.01 0.10 679 227 6.98 20,433 1.18 0.57 −52 228 5.28 19,348 0.14 0.03−81 229 5.31 18,787 0.10 0.05 −49 230 5.93 18,712 0.76 0.00 −100 2315.64 18,600 0.31 0.22 −28 232 6.67 19,523 0.14 0.11 −20 233 8.59 18,5751.65 5.90 259 234 5.07 17,292 2.11 0.73 −66 235 6 18,046 0.00 0.01 6403236 8.95 18,029 0.11 0.05 −58 237 5.4 17,776 0.49 0.00 −99 238 5.2117,627 0.01 0.15 1079 239 4.96 16,512 1.00 0.17 −83 240 8.79 17,586 0.100.65 562 241 6.55 17,843 0.05 0.01 −87 242 6.69 17,703 0.03 0.11 222 2436.83 17,213 0.10 0.15 59 244 8.68 16,051 1.61 0.01 −99 245 7.4 16,8970.02 0.21 824 246 6.25 15,855 0.27 0.10 −64 247 6.23 15,342 0.25 0.71180 248 7.25 16,345 0.05 0.06 12 249 6.04 15,269 0.01 0.21 2260 250 7.1115,932 0.07 0.03 −61 251 nd nd 0.26 1.52 496 252 6.69 14,760 0.22 0.51136 253 7.32 14,729 2.34 0.82 −65 254 nd nd 0.07 0.46 598 255 nd nd 1.390.03 −98 nd = not determined; ++++ = greater than 200,000

Based on the number of differences in the 2D gels for HVF and HVC virionparticle polypeptide analyses and the different size and morphology ofthe HVF virion particles shown in FIG. 13 (as compared to particlesproduced using helper virus), it is clear the HSV amplicon particlesproduced according to the present invention are different in kind fromthe HSV amplicon particles produced using a helper virus in accordancewith previously known techniques.

Although the invention has been described in detail for purposes ofillustration, it is to be understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method for producing herpes simplex virus (HSV) amplicon particles,comprising: co-transfecting a host cell with the following: (i) anamplicon vector comprising an HSV origin of replication, an HSVcleavage/packaging signal, and a heterologous transgene expressible in apatient, (ii) one or more vectors individually or collectively encodingall essential HSV genes but excluding all cleavage/packaging signals,and (iii) a vhs expression vector encoding a virion host shutoffprotein; and isolating HSV amplicon particles produced by the host cell,the HSV amplicon particles including the transgene.
 2. The methodaccording to claim 1, wherein the isolated HSV amplicon particles aresubstantially pure.
 3. The method according to claim 1, wherein thevirion host shutoff protein is selected from the group consisting ofHSV-1 virion host shutoff protein, HSV-2 virion host shutoff protein,HSV-3 virion host shutoff protein, bovine herpesvirus 1 virion hostshutoff protein, bovine herpesvirus 1.1 virion host shutoff protein,gallid herpesvirus 1 virion host shutoff protein, gallid herpesvirus 2virion host shutoff protein, suid herpesvirus 1 virion host shutoffprotein, baboon herpesvirus 2 virion host shutoff protein, pseudorabiesvirus virion host shutoff protein, cercopithecine herpesvirus 7 virionhost shutoff protein, meleagrid herpesvirus 1 virion host shutoffprotein, equine herpesvirus 1 virion host shutoff protein, and equineherpesvirus 4 virion host shutoff protein.
 4. The method according toclaim 3, wherein the virion host shutoff protein is selected from thegroup consisting of HSV-1 virion host shutoff protein, HSV-2 virion hostshutoff protein, and HSV-3 virion host shutoff protein.
 5. The methodaccording to claim 4, wherein the vhs expression vector comprises: a DNAmolecule encoding the HSV virion host shutoff protein operativelycoupled to its native transcriptional control elements.
 6. The methodaccording to claim 1, wherein the vhs expression vector comprises: a DNAmolecule encoding the virion host shutoff protein; a promoter elementoperatively coupled 5′ to the DNA molecule; and a transcriptiontermination element operatively coupled 3′ to the DNA molecule.
 7. Themethod according to claim 1, wherein the host cell expresses a VP16protein.
 8. The method according to claim 7, wherein the VP16 protein isselected from the group consisting of HSV-1 VP16, HSV-2 VP16, bovineherpesvirus 1 VP16, bovine herpesvirus 1.1 VP16, gallid herpesvirus 1VP16, gallid herpesvirus 2 VP16, meleagrid herpesvirus 1 VP16, andequine herpesvirus 4 VP16.
 9. The method according to claim 7 furthercomprising: transfecting the host cell, prior to said co-transfecting,with a vector encoding the VP16 protein.
 10. The method according toclaim 9, wherein said transfecting is carried out at least about 4 hoursprior to said co-transfecting.
 11. The method according to claim 7,wherein the host cell stably expresses the VP16 protein.
 12. The methodaccording to claim 1, wherein the isolated HSV amplicon particles arepresent at a concentration of greater than 1×10⁶ particles permilliliter.
 13. The method according to claim 1 further comprising:concentrating the isolated HSV amplicon particles to a concentration ofat least about 1 ×10⁷ particles per milliliter.
 14. The method accordingto claim 1 wherein the transgene encodes a therapeutic transgeneproduct.
 15. The method according to claim 14, wherein the therapeutictransgene product is a protein or an RNA molecule.
 16. The methodaccording to claim 15, wherein the therapeutic transgene product is anRNA molecule selected from the group consisting of antisense RNA, aninhibitory RNA, and an RNA ribozyme.
 17. The method according to claim15, wherein the therapeutic transgene product is a protein selected fromthe group consisting of receptors, signaling molecules, transcriptionfactors, growth factors, apoptosis inhibitors, apoptosis promoters, DNAreplication factors, enzymes, structural proteins, neural proteins, andhistone or non-histone proteins.
 18. An HSV amplicon particle producedaccording to the process of claim
 1. 19. An HSV amplicon particleproduced according to the process of claim
 14. 20. A kit for preparingHSV amplicon particles comprising: an amplicon vector comprising an HSVorigin of replication, an HSV cleavage/packaging signal, and a transgeneinsertion site; one or more vectors individually or collectivelyencoding all essential HSV genes but excluding all cleavage/packagingsignals; a vhs expression vector encoding an virion host shutoffprotein; a population of host cells susceptible to transfection by theamplicon vector, the vhs expression vector, and the one or more vectors;and directions for transfecting the host cells under conditions toproduce HSV amplicon particles.
 21. The kit according to claim 20further comprising: a vector encoding a VP16 protein.
 22. The kitaccording to claim 21, wherein the VP16 protein is selected from thegroup consisting of HSV-1 VP16, HSV-2 VP16, bovine herpesvirus 1 VP16,bovine herpesvirus 1.1 VP16, gallid herpesvirus 1 VP16, gallidherpesvirus 2 VP16, meleagrid herpesvirus 1 VP16, and equine herpesvirus4 VP16.
 23. The kit according to claim 20, wherein the host cell stablyexpresses a VP 16 protein.
 24. The kit according to claim 23, whereinthe VP16 protein is selected from the group consisting of HSV-1 VP16,HSV-2 VP16, bovine herpesvirus 1 VP16, bovine herpesvirus 1.1 VP16,gallid herpesvirus 1 VP16, gallid herpesvirus 2 VP16, meleagridherpesvirus 1 VP16, and equine herpesvirus 4 VP16.
 25. The kit accordingto claim 20, wherein the virion host shutoff protein is selected fromthe group consisting of HSV-1 virion host shutoff protein, HSV-2 virionhost shutoff protein, HSV-3 virion host shutoff protein, bovineherpesvirus 1 virion host shutoff protein, bovine herpesvirus 1.1 virionhost shutoff protein, gallid herpesvirus 1 virion host shutoff protein,gallid herpesvirus 2 virion host shutoff protein, suid herpesvirus 1virion host shutoff protein, baboon herpesvirus 2 virion host shutoffprotein, pseudorabies virus virion host shutoff protein, cercopithecineherpesvinis 7 virion host shutoff protein, meleagrid herpesvirus 1virion host shutoff protein, equine herpesvirus 1 virion host shutoffprotein, and equine herpesvirus 4 virion host shutoff protein.
 26. Thekit according to claim 25, wherein the virion host shutoff protein isselected from the group consisting of HSV-1 virion host shutoff protein,HSV-2 virion host shutoff protein, and HSV-3 virion host shutoffprotein.
 27. The kit according to claim 26, wherein the vhs expressionvector comprises: a DNA molecule encoding the HSV virion host shutoffprotein operatively coupled to its native transcriptional controlelements.
 28. The kit according to claim 20, wherein the vhs expressionvector comprises: a DNA molecule encoding the virion host shutoffprotein; a promoter element operatively coupled 5′ to the DNA molecule;and a transcription termination element operatively coupled 3′ to theDNA molecule.